Methods of producing influenza vaccine compositions

ABSTRACT

Methods and compositions for the optimization of production of influenza viruses suitable as influenza vaccines are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/450,181 filed Feb. 25, 2003, entitled “METHODS OF PRODUCING INFLUENZAVACCINE COMPOSITIONS.” This prior application is hereby incorporated byreference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

Vaccines against various and evolving strains of influenza are importantnot only from a community health stand point, but also commercially,since each year numerous individuals are infected with different strainsand types of influenza virus. Infants, the elderly, those withoutadequate health care and immuno-compromised persons are at special riskof death from such infections. Compounding the problem of influenzainfections is that novel influenza strains evolve readily, therebynecessitating the continuous production of new vaccines.

Numerous vaccines capable of producing a protective immune responsespecific for such different influenza viruses have been produced forover 50 years and include, e.g., whole virus vaccines, split virusvaccines, surface antigen vaccines and live attenuated virus vaccines.However, while appropriate formulations of any of these vaccine types iscapable of producing a systemic immune response, live attenuated virusvaccines have the advantage of being also able to stimulate localmucosal immunity in the respiratory tract. A vaccine comprising a liveattenuated virus that is also capable of being quickly and economicallyproduced and that is capable of easy storage/transport is thus quitedesirable.

To date, all commercially available influenza vaccines have beenpropagated in embryonated hen eggs. Although influenza virus grows wellin hen eggs, the production of vaccine is dependent on the availabilityof such eggs. Because the supply of eggs must be organized, and strainsfor vaccine production selected months in advance of the next fluseason, the flexibility of this approach can be limited, and oftenresults in delays and shortages in production and distribution.Therefore, any methods to increase throughput and/or increase output ofvaccine production in hen eggs is greatly desirable.

Systems for producing influenza viruses in cell culture have also beendeveloped in recent years (See, e.g., Furminger. Vaccine Production, inNicholson et al. (eds.) Textbook of Influenza pp. 324–332; Merten et al.(1996) Production of influenza virus in cell cultures for vaccinepreparation, in Cohen & Shafferman (eds.) Novel Strategies in Design andProduction of Vaccines pp. 141–151). While eliminating many of thedifficulties related to vaccine production in hen eggs, not allpathogenic strains of influenza grow well in cell culture, or can beproduced according to established tissue culture methods. In addition,many strains with desirable characteristics, e.g., attenuation,temperature sensitivity and cold adaptation, suitable for production oflive attenuated vaccines, have not been successfully grown in tissueculture using established methods. Therefore, any methods to increasethroughput and/or increase output of vaccine production in cell cultureis also greatly desirable.

Considerable work in the production of influenza virus for production ofvaccines has been done by the present inventors and co-workers; see,e.g., Multi-Plasmid System for the Production of Influenza Virus, U.S.Ser. No. 60/375,675 filed Apr. 26, 2002, PCT/US03/12728 filed Apr. 25,2003 and U.S. Ser. No. 10/423,828 filed Apr. 25, 2003, etc. The presentinvention provides methods of increasing/optimizing production (in bothquantity/quality and speed) of such viruses, as well as for otherinfluenza viruses, for production of vaccine compositions. Aspects ofthe current invention are applicable to traditional hen egg and new cellculture vaccine production styles (and also combined systems) andcomprise numerous other benefits that will become apparent upon reviewof the following.

SUMMARY OF THE INVENTION

The invention provides embodiments of methods of making one or moreinfluenza virus compositions by passaging an influenza virus (e.g., an Avirus strain or a B virus strain, etc.) through eggs, heating the virusand filtering the virus through a membrane. In some such embodiments,the filtering comprises passage of the composition through a microfilterof a pore size ranging from 0.2 micrometers to about 0.45 micrometers.Furthermore, in various embodiments, the temperature of heating in suchembodiments optionally comprises from about 28° C. to about 40° C. ormore, while in some embodiments, the temperature comprises 31° C. orfrom about 30° C. to about 32° C. The heating in such embodimentsoptionally occurs before or during or before and during the filtrationand optionally comprises from about 50 minutes to about 100 minutes,from about 60 minutes to about 90 minutes, or about 60 minutes. Theinvention also provides an influenza virus composition produced by suchmethods (including wherein the composition is a vaccine composition).

In other aspects, the invention comprises a method of making one or moreinfluenza virus composition by passaging an influenza virus througheggs, heating the virus, and purifying the virus. Such embodiments alsooptionally include filtering the composition through a membrane andwherein the compositions comprises a vaccine composition as well as theactual vaccine composition produced by such embodiment.

In related aspects, the invention comprises a method of making one ormore influenza virus composition, by passaging an influenza virusthrough eggs which are rocked during the passage. The rocking optionallycomprises tilting the eggs at a rate of about 1 cycle per minuteoptionally for about 12 hours. Such embodiments optionally use influenzaA virus strains and/or influenza B virus strains and also optionallycomprise wherein a TCID₅₀ of such rocked eggs is 0.4 log greater than aTCID₅₀ of the same virus passaged through non-rocked eggs. Viruscompositions produced by such embodiments are also features of theinvention, including wherein the compositions are vaccine compositions.

The invention also comprises methods of making one or more influenzavirus composition (e.g., biasing the reassortment of such) byintroducing a plurality of vectors comprising an influenza virus genomeinto a population of host eggs (which are capable of supportingreplication of the virus), culturing the population of eggs at atemperature less than or equal to 35° C., and recovering a plurality ofinfluenza viruses. Such viruses optionally comprise, e.g., an attenuatedvirus, a cold adapted virus, a temperature sensitive virus or anattenuated cold adapted temperature sensitive virus, and can alsocomprise, e.g., an influenza B virus. A virus composition produced bysuch an embodiment is also a feature of the invention (including vaccinecompositions). Such aspects also optionally include further selectingfor influenza viruses containing wild-type HA and NA genes (e.g., byincubating the plurality of viruses with one or more antibodies specificfor non-wild-type HA and NA genes (e.g., done within the one or moreegg). Virus compositions produced thusly are also features of theinvention, including vaccine compositions.

Other aspects of the invention include making one or more influenzavirus composition by introducing a plurality of vectors comprising aninfluenza virus genome into a population of host eggs (which is capableof supporting replication of influenza virus), culturing the populationof eggs at a temperature less than or equal to 35° C., recovering aplurality of viruses, incubating the plurality of viruses with one ormore antibodies specific for non-wild-types HA and NA genes, passagingthe virus through eggs (which are rocked) and heating the virus andfiltering the virus through a membrane. Viruses produced by such methodsare also features of the invention (including vaccine compositions).

In the various methods embodied herein, the influenza virus compositionis optionally assayed through use of a fluorescence focus assay. Suchvirus compositions optionally comprise from about 10% to about 60%unfractionated normal allantoic fluid (and optionally from about 1% toabout 5% arginine). The compositions are optionally diluted with abuffer which is optionally substantially free of normal allantoic fluid.The compositions herein are optionally substantially free of gelatin.These compositions are stable from about 2° C. to about 8° C., or arestable at 4° C. In some compositions and methods herein, the viruses areinfluenza viruses, while in yet other compositions and methods herein(e.g., those involving microfiltration and/or ultrafiltration and/orheating and/or rocking) the viruses optionally comprise, e.g.,non-influenza viruses (e.g., viruses that are produced through culturein eggs, e.g., myxoviruses, paramyxovirus, RSV, mumps virus, measlesvirus, Sendi virus, yellow fever virus, pIV, etc.). Thus, the methodsand compositions of the invention are also applicable to such otherviruses and/or to non-influenza viruses.

In yet other aspects, the invention comprises an influenza viruscomposition, wherein the composition is made by: passaging the influenzavirus through eggs, heating the virus, and filtering the virus through amembrane, which composition has a first TCID₅₀, which first TCID₅₀ isgreater than a second TCID₅₀, which second TCID₅₀ results from aninfluenza virus not made by: passaging the virus through eggs, heatingthe virus, and filtering the virus through a membrane.

Other aspects of the invention include an influenza virus composition,wherein the composition is made by: passaging the influenza virusthrough eggs, wherein the eggs are rocked during said passage, whichcomposition has a first TCID₅₀, which first TCID₅₀ is greater than asecond TCID₅₀, which second TCID₅₀ results from an influenza virus notmade by: passaging the influenza virus through eggs, wherein the eggsare rocked during said passage.

Still other embodiments herein include an influenza virus composition,wherein the composition is made by: introducing a plurality of vectorscomprising an influenza virus genome into a population of host eggs,which population of host eggs is capable of supporting replication ofinfluenza virus, culturing the population of host eggs at a temperatureless than or equal to 35° C., and recovering a plurality of influenzaviruses, which composition has a first TCID₅₀, which first TCID₅₀ isgreater than a second TCID₅₀, which second TCID₅₀ results from aninfluenza virus not made by: introducing a plurality vectors comprisingan influenza virus genome into a population of host eggs, whichpopulation of host eggs is capable of supporting replication ofinfluenza virus, culturing the population of host eggs at a temperatureless than or equal to 35° C., and recovering a plurality of influenzaviruses.

These and other objects and features of the invention will become morefully apparent when the following detailed description is read inconjunction with the accompanying figures appendix.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Displays M Genotyping after infection at 33° C. and 25° C. inDEK TC-24.

FIG. 2: Displays a plaque assay, and data, showing the different titersof 5:3 and 6:2 at 33° C.

FIG. 3: Displays growth curves of 6:2 vs. 5:3 reassortants.

FIG. 4: Displays the M1 sequences of MDV-B and wild-type B viruses(wt_B_Yamanashi_M1, SEQ ID NO:1; wt_B_JHG_(—)5_(—)99_M1, SEQ ID NO:2;wt_B_Vic_(—)504_(—)2000_M1, SEQ ID NO:3; MDV-B-M1, SEQ ID NO:4; andwt_B_HK_(—)330_01_M1, SEQ ID NO:5).

FIG. 5: Displays the M2 sequences of MDV-B and wild-type B viruses(MDV-B-M2, SEQ ID NO:6; wt_B_HK_(—)330_(—)01_M2, SEQ ID NO:7;wt_B_JHG_(—)5_(—)99_M2, SEQ ID NO:8; wt_B_Yamanashi_M2, SEQ ID NO:9; andwt_B_Vic_(—)504_(—)2000_M2, SEQ ID NO:10).

FIG. 6: Displays mutations on the two conservative sites in MDV B-M1.

FIG. 7: Displays the growth curves of the B/HK 6:2 M1 mutations.

FIG. 8: Shows various CEK cell infections at different MOIs.

FIG. 9: Shows a flow chart of potential microbial contamination duringvaccine production process.

FIG. 10: Illustrates temperature decay rates of individual eggs viainfra-red imaging.

FIG. 11: Illustrates thermal imaging of live, infertile, and dead eggs.

FIG. 12: Illustrates a schematic flowchart of virus harvestconcentration.

FIG. 13: Displays comparison of the 5^(th) wash with NAF proteins.

FIG. 14: Displays an assay of an N/New Caledonia/20/99 1-×-Neat samplebefore concentration.

FIG. 15: Displays an assay of an A/New Caledonia/20/99 10× concentratedsample.

FIG. 16, Panels A–B: Display a comparison of 1× and 10× of A/NewCaledonia/20/99; and 1×-W sample after 5 washes.

FIG. 17: Displays a comparison of A/New Caledonia/20/99 1× and 1×-Wsamples.

FIG. 18, Panels A–C: Display a comparison of 10× and 10×-W of A/NewCaledonia/20/99; Permeate of A/New Caledonia/20/99; and 5 washes ofA/New Caledonia/20/99.

FIG. 19: Shows an analysis by SEC comparing times washed and impuritiesremoved.

FIG. 20: Displays 1×W and 10×-W comparison of A/New Caledonia/20/99.

FIG. 21: Shows a 96-well plate assay of A/New Caledonia/20/99.

FIG. 22: Shows a graph neuraminidase activity/virus purification inretentate and permeate.

FIG. 23: Displays RHPLC of Control, 10×, 10×-W, and 1×-W.

FIG. 24: Shows a graph of Control, 10×, 1×-W and 10×-w samples.

FIG. 25: Displays RHPLC of permeate and washes 1 to 6.

FIG. 26: Shows a graph of RHPLC ovomucoid removal (peak area).

FIG. 27: Shows a graph of RHPLC of lysozyme removal (peak area).

FIG. 28: Shows a graph of RHPLC of conalbumin removal (peak area).

FIG. 29: Shows a graph of RHPLC of ovalbumin removal (by peak area).

FIG. 30: Shows a graph of ovalbumin analysis by Agilent 2100.

FIG. 31: Is a Western blot SDS-PAGE gel of anti-A/New Caledonia.

FIG. 32: Displays assays of 10×-W, sample after 5 washes of A/NewCaledonia/20/99.

FIG. 33: Shows a graph of RNA analysis by RTPCR.

FIG. 34: Shows monitoring of A/Beijing—cell culture propagation by SEC.

FIG. 35: Shows cell culture harvest of A/Beijing in Vero cells.

FIG. 36: Shows concentration of 2 liters of A/Panama cell culture.

FIG. 37: Shows concentration of 2 liters of B/Hong Kong cell culturedown to 10 ml.

FIG. 38: Shows 4 graphs of stability of exemplary virus storageformulations.

FIG. 39: Shows a graph of stability of exemplary virus storageformulations with various virus strains.

FIG. 40: Shows a graph of stability of exemplary virus storageformulations with various virus strains.

FIG. 41, Panels A–C: Show stability of exemplary virus storageformulations with differing citrate concentrations.

FIG. 42, Panels A–C: Show stability of exemplary virus storageformulations with differing EDTA concentrations.

FIG. 43: Displays stability of unpurified virus harvest formulationswith different virus strains over 9 months.

FIG. 44: Illustrates initial potency loss of formulations associatedwith phosphate buffer.

FIG. 45: Gives a global picture of stability slopes of variousformulations at 6 months.

FIG. 46: Gives a global picture of stability slopes of variousformulations at 6 months.

FIG. 47: Shows stability of various formulations with gelatin andPVP/EDTA.

FIG. 48: Shows stability of various formulations with histidine atdifferent pH.

FIG. 49: Shows stability of various formulations with different amountsof sucrose.

FIG. 50: Histogram derived from plotting the absorbance readings fromthe wells, versus the frequency of the values (number of wells read atthat absorbance value).

FIG. 51: Histogram derived from plotting absorbance readings versusfrequency of values.

FIG. 52: Shows a generic process for generating 6:2 influenzareassortants.

DETAILED DESCRIPTION

The present invention includes methods and compositions to increasethroughput and output of viruses and virus composition productionssuitable for vaccine production/use. Included are methods andcompositions for, e.g., selecting for desired reassortants in virusproduction, temperature conditioning/filtration, rocking, antibodyselection, potency assays, and many additional features as described inmore detail herein.

It will be appreciated by those skilled in the art that the varioussteps herein are not required to be all performed or exist in the sameproduction series. Thus, while in some preferred embodiments, all stepsand/or compositions herein are performed or exist, e.g., as outlined inTable 1, in other embodiments, one or more steps are optionally, e.g.,omitted, changed (in scope, order, placement, etc.) or the like.

The basic overview of the methods and compositions for virus productionherein are outlined in Table 1. Once again, as is to be emphasizedthroughout, the individual steps of the invention, such as those listedin Table 1 are not necessarily mutually dependent. For example, in someembodiments, eggs containing an appropriate reasserted virus solutionare rocked during incubation (see below), while in other embodimentsthey are not; heating and filtration in Step 10 is independent of use ofuniversal reagents in Step 13, etc. The presence of any onestep/method/composition in the invention is not dependent upon thenecessary presence of any other step/method/composition in theinvention. Therefore, various embodiments of the current invention caninclude only one of the steps, all of the steps, or any and all variouscombinations of the steps.

It will also be appreciated by those skilled in the art that typicalembodiments comprise steps/methods/compositions that are known in theart, e.g., candling of virus containing eggs, inoculation of eggs withviruses, etc. Therefore, those skilled in the art are easily able todetermine appropriate conditions, sub-steps, step details, etc., forsuch known steps to produce the appropriate viruses, virus solutions,compositions, etc. The individual steps are described in greater detailbelow. See Table 1 for listing of major steps involved in exampleembodiment.

For ease in discussion and description, the various steps of the currentinvention, e.g., the various methods and compositions, can be thought ofas comprising or falling into four broad groups. The first groupcomprises such aspects as co-infection, reassortment, selection ofreassortants, and cloning of reassortants (e.g., thereby roughlycorresponding to Steps 1 through 3 in Table 1). The second groupcomprises such aspects as purification and expansion of reassortants andcan be thought of as roughly corresponding to Steps 4 through 6 inTable 1. The third group comprises further expansion of reassortants ineggs, along with harvesting and purification of such harvested virussolutions (e.g., roughly corresponding to Steps 7 through 11 in Table1). The fourth group comprises stabilization of harvested virussolutions and potency/sterility assays of the virus solutions (e.g.,roughly corresponding to Steps 12 through 15 in Table 1). It is to beunderstood, however, that division of the aspects of the invention intothe above four general categories is solely forexplanatory/organizational purposes and no inference of interdependenceof steps, etc. should be made.

DETAILED DESCRIPTION OF STEPS

As mentioned above, for ease in discussion and description, the varioussteps of the current invention can be thought of as comprising fourbroad groups. The first group comprises such aspects as co-infection,reassortment, selection of reassortants, and cloning of reassortants(e.g., thereby roughly corresponding to Steps 1 through 3 in Table 1).The second group comprises such aspects as purification and expansion ofreassortants and can be thought of as roughly corresponding to Steps 4through 6 in Table 1. The third group comprises further expansion ofreassortants in eggs, along with harvesting and purification of suchharvested virus solutions (e.g., roughly corresponding to Steps 7through 11 in Table 1). The fourth group comprises stabilization ofharvested virus solutions and potency/sterility assays of the virussolutions (e.g., roughly corresponding to Steps 12 through 15 in Table1). It is to be emphasized, however, that division of the aspects of theinvention into the above four general categories is solely forexplanatory/organizational purposes and no inference of interdependenceof steps, etc. should be made.

Group 1

The aspects of the current invention which are broadly classified hereinas belonging to Group 1, comprise methods and compositions related tooptimization of co-infection of cell culture lines, e.g., with a masterdonor virus and one or more wild-type viruses in order to producespecifically desired reasserted viruses; selection of appropriatereassorted viruses; and cloning of the selected reassorted viruses.Reassortment of influenza virus strains is well known to those of skillin the art. Reassortment of both influenza A virus and influenza B virushas been used both in cell culture and in eggs to produce reassertedvirus strains. See, e.g., Tannock et al., Preparation andcharacterisation of attenuated cold-adapted influenza A reassortantsderived from the A/Leningrad/134/17/57 donor strain, Vaccine (2002)20:2082–2090. Reassortment of influenza strains has also been shown withplasmid constructs. See, “Multi-Plasmid System for the Production ofInfluenza Virus,” cited above.

Reassortment, in brief, generally comprises mixing (e.g., in eggs orcell culture) of gene segments from different viruses. For example, thetypical 8 segments of influenza B virus strains can be mixed between,e.g., a wild-type strain having an epitope of interest and a “donor”strain, e.g., comprising a cold-adapted strain. Reassortment between thetwo virus types can produce, inter alia, viruses comprising thewild-type epitope strain for one segment, and the cold-adapted strainfor the other segments. Unfortunately, to create the desiredreassortants, a sometimes large number of reassortments need to be done.After being reassorted, the viruses can also be selected (e.g., to findthe desired reassortants). The desired reassortants can then be cloned(e.g., expanded in number). Steps to decrease the time required forconstruction of reassortants and to enhance creation of desiredreassortants are, thus, highly desirable.

Traditional optimization, selection, and cloning of desired reassortantsfor influenza B virus, typically occurs by co-infection of virus strainsinto a cell culture (e.g., CEK cells) followed by selection withappropriate antibodies, e.g., against material from one of the parentvirus, (usually done in eggs), and cloning or expanding of virus, etc.which is typically done in cell culture. However, such traditionalreassortment presents drawbacks in that thousands of reassortments areneeded to create the desired segment mix. When such reassortments aredone, it is apparent that truly random reassortments are not the endresult. In other words, pressures that bias the process exist in thesystems. For influenza A strains, however, such processes do not appearto have such bias. For A strains, co-infection of strains (typicallyinto cell culture such as CEK cells) is followed by selection andcloning at the same time, again, typically in cell culture.

Thus, as detailed herein, various embodiments of the invention comprisesteps to reduce the reassortment bias. Namely, cloning of reassortantsis done in eggs (e.g., at 33° C.) rather than in cell lines, or is donein cell lines, but at, e.g., 25° C.

Optimization of Reassortment

The current invention utilizes the steps in Group 1 to optimize thereassortment process in order to reduce the number of reassortmentsneeded (and thus increase the throughput of the vaccine productionprocess). The steps utilizing such optimization techniques are typicallyembodied with reassortment of influenza B strains and are typically donein cell culture, e.g., CEK cells.

Other methods of reassortment of influenza virus mix dilutions of amaster donor virus (MDV) and a wild-type virus, e.g., a 1:5 dilution ofeach no matter the concentration of the respective solutions, which arethen incubated for 24 and 48 hours at 25° C. and 33° C. However, whilesuch an approach is often acceptable for influenza A strains, influenzaB strains do not typically give positive results with such protocol. Forexample, to achieve the proper 6:2 assortment (i.e., 6 genes from theMDV and 2 genes, NA and HA from the wild-type virus) thousands ofreassortments must often be done.

Thus, typical embodiments of the steps in Group 1 of the inventioncomprise determination of the MOI (multiplicity of infection) of the MDVstrain and the wild-type strains (especially for influenza B strainsused), followed by reassortments comprising those illustrated in Table2. The incubations of such optimized reassortment mixtures is carriedout at 33° C. for 24 hours in eggs. In embodiments like this, proper 6:2reassortments are typically achieved by screening hundreds ofreassortment mixes as opposed to thousands of reassortment mixesnecessary in non-optimized systems.

Selection and Cloning of Reassortments

The steps in Group 1 also comprise selection of reasserted influenzaviruses. The methods and compositions of the current invention areespecially useful for (and are typically embodied for) selection ofproperly reasserted influenza B viruses. Reassorted influenza A strainsare capable of selection in either cell culture (e.g., CEK cells) or ineggs. However, reasserted influenza B strains present problems whenreassorted in cell culture (e.g., when selected for in CEK cells). It isbelieved that CEK cells interfere with the M gene in influenza Bstrains, thus reducing the overall production. See below. The currentinvention takes notice of such suppression by, in some embodiments,having selection of influenza B reassortments done in eggs (which areneutral in terms of selection pressure against the M gene in influenza Bstrains) at 33° C. or, alternatively, in CEK cells at 25° C. See FIG.52.

Other embodiments of the invention in Group 1 include use of anti-HA (ofthe MDV) and anti-NA (of the MDV) antisera in the selection process,thus, achieving a stronger selection.

Yet other embodiments of the invention in Group 1 include cloning of thereassortments produced. As will be apparent from the previousdiscussion, cloning out of influenza B reassortments in CEK cell culturehas proven problematic because of negative selection pressure. Thus, insome embodiments herein, B strain reassortments are cloned out in eggsat 33° C. A strain reassortments, on the other hand, are optionallycloned out and selected at the same time in CEK cell culture.

Even though some embodiments herein take advantage of the non-bias ornon-suppression of eggs on reassortments (see above), other embodimentsherein comprise selection/cloning of reassortments in cell culture, butat 25° C. Thus, some aspects of the current invention compriseembodiments which take advantage of the different properties of MDVB(master donor virus B) M gene and wild-type B virus M gene. For example,6:2 and 5:3 co-infections are optionally done to produce the desiredreassortments. Thus, for example, in the B/HongKong/330/01 MVSproduction process, the cloning from mixed wild-type and cold-adapted Mviral RNAs by limiting dilution in CEK cells at 33° C., results in thedominant growth of wild-type M viral RNA. In both eggs and CEK cells,the wild-type M vRNA is dominant over MDV-derived M vRNA when 6:2 iscoinfected with .5:3 (containing wild-type M gene) at 33° C., althoughthe chance of getting wild-type M vRNA in eggs is higher. In contrast,both MDV-derived and wild-type M vRNAs are present in comparable amountwhen 6:2 and 5:3 are coinfected into CEK cells at 25° C. Therefore, insome embodiments herein 25° C. is used for 6:2 cloning in CEK cells inthe MVS process. See, FIGS. 1 through 8. From the Figures it can be seenthat plaque assays show that the titer of B virus 6:2 at 33° C. is atleast 2 log10 lower than respective 5:3 at low MOI, while 6:2 grows tothe same level as 5:3 at 25° C. The growth defect of 6:2 at 33° C. mayaccount for the selection against 6:2 in MVS CEK cloning. The differentgrowth properties of MDVB and 6:2 suggest the involvement of HA, NA inthe M gene dominance. There are only two conservative amino aciddifferences between MDVB and wild-type B viruses. A single mutation ofValine to wild-type conservative Methionine on the 6:2 M1 gene is ableto reverse the growth defect of 6:2 in CEK cells at 33° C.

Characterization of Reassortments

Yet other embodiments of the current invention utilize applications of ahigh throughput single strand conformation polymorphism/capillaryelectrophoresis (SSCP/CE) assay to determine the gene constellation ofinfluenza viruses used herein. It should be appreciated that suchcharacterization aspect can also be classified into other “Groups”herein, but is discussed here for organizational purposes. Influenzaviruses contain 8 gene segments and, as described above, co-infection ofa single cell with two different influenza strains can producereassortant viruses with novel gene constellations distinct from eitherparent. Thus, some embodiments herein use a SSCP/CE assay to rapidlydetermine the gene segment constellation of a large number of influenzavirus samples. The influenza viral gene segments are optionallyamplified by RT-PCR using fluorescent-labeled primers specific for eachof the eight segments. See, also, Arvin et al. (2000) J. Clin. Micro.38(2):839–845 which is incorporated herein by reference for allpurposes.

In order to reduce the number of RT-PCR reactions required to genotypeall eight segments of the influenza genome, a multiplex reaction isoptionally created in which multiple segments are simultaneouslyamplified in the same reaction. The RT-PCR products corresponding toeach segment are differentiated by size, migration pattern andfluorescent color. The migration of a single strand DNA fragment in anon-denaturing matrix is determined not only by its size but also by itssequence content.

Cells are optionally co-infected with cold-adapted B/Ann Arbor/1/66(MDV-B) or similar, and one of several wild-type influenza B strains.The progeny of the co-infection are cloned by limiting dilution and thenucleic acids amplified in multiplex reactions. Primers are selected andproducts separated by SSCP/CE at 18° C., which enhances the resolutionbetween MDV B and wild-type strains' eight gene segments.

For example, to demonstrate the accuracy of the SSCP/CE assay, 400 genesegments from approximately 50 different reassortant viruses wereanalyzed and the SSCP/CE results were compared to those obtained byrestriction fragment length polymorphism (RFLP). It was found that therewas a high concordance (˜98%) between the two sets of data, therebyvalidating the SSCP/CE assay. Furthermore, it was shown that the SSCP/CEassay was capable of detecting a single nucleotide substitution withinthe M gene segment of influenza B virus.

Prevention of Bacterial Contamination

Some embodiments of the current invention comprise steps to detectand/or prevent/detect microbial contamination of eggs in which influenzavirus is produced. Such steps are useful in several areas as outlined inTable 1 and can be included in Groups 1, 2, and 3, but fororganizational purposes are presented with the steps of Group 1. Themicrobial detection strategies of the invention are useful forrapid/high throughput microbial detection and, thus, as with many othersteps herein, are useful for increasing throughput in virus/vaccineproduction.

Many current influenza vaccine production strategies, including someembodiments of the invention herein, use as a component, the traditionalmethod for influenza virus expansion in specific-pathogen-free fertilechicken eggs. Possible microbial contamination can occur in severalpoints in the production of virus in eggs. See, e.g., FIG. 9, whichoutlines one possible example of a virus production flowchart andpossible areas of contamination therein. Unfortunately, the chicken eggsmay have some microorganisms outside of their shells as part of theirnatural flora. It is also possible to have microorganisms enclosedwithin the shell of the egg during the development of the chickenembryo. Fertilized chicken eggs are incubated at 37° C. in high humidityfor development of the embryo, which constitutes prime incubationconditions for many types of microbial contaminants as well. Anotherpossible time of microbial contamination occurs when the shell ispunctured to inoculate the egg. Even though prior to virus inoculation,the eggs are often sprayed with alcohol, there is still opportunity formicroorganisms to enter into the egg.

After expansion of viruses for 2 to 3 days in the eggs, the top of theegg shell is typically removed for manual harvesting of the allantoicfluid containing virus within the egg. This harvesting is another pointwhere microbial contamination may originate. Unfortunately eggs withsuch contaminating bioburden may escape detection, necessitating poolinginto multiple bottles to minimize the rejection of the entire lot due toa failed MPA test. Since three influenza strains are typically used invaccine production, blending of the three strains is required for thefinal bulk. In-process MPA (microbiological purity assay) testing isperformed, e.g., at virus harvest (see FIG. 9) prior to use in theblending and filling to ensure microbial-free product.

After incubation, the “traditional” method of candling is used toidentify infertile and dead eggs which are possibly dead due to naturalcauses or to microbial contamination (i.e., dead eggs may occur due toinfectivity of the virus and/or expansion of microorganisms, both ofwhich require detection and removal of such eggs). Candling comprises,e.g., the process of holding an egg in front of a light source in adarkened room to enable visualization of the developing embryo. Deadeggs are excluded from virus inoculation.

As can be seen from the above points, detection of microbialcontamination can be needed at multiple steps during the manufacture ofinfluenza vaccine. There is a need to eliminate or reduce avian andenvironmental microbes and a need to eliminate or reduce introduction ofenvironmental and human microbes. Thus, a need for non-invasive andrapid methods of screening eggs to identify and remove infertile, dead,or microbially contaminated eggs exists. Such methods should preferablybe non-invasive and rapid. Current methods for detection ofcontaminating microorganisms include, e.g., compendial methods (MPA andBioburden). Current methods can include, e.g., egg candling during eggpre/post inoculation (which is typically done manually at a rate ofabout 500 eggs/hour/person); MPA and BioBurden tests which are typicallymanual and take about 14 days for MPA and about 3 days for BioBurden(which are done during virus harvest); mycoplasma testing; which istypically done manually and takes about 28 days (done during virusharvest); and mycobacterium testing which is typically manual and takesabout 56 days (done during virus harvest). From such, it will beappreciated that there are opportunities for significant reduction inturn around times for the traditional methods. New methods arepreferable, e.g., to reduce time to result from days to 24 hours or less(and preferably 4 hours or less for in-process testing) and from weeksto a few days for Release Testing. Other preferences include, e.g., toreduce to intermediate/inventory hold-time, to potentially expediteproduct release/approval, and to reduce cost/labor/overhead. In general,any method chosen to detect microbial contamination should consider,e.g., scientific requirements such as intended use, time to result,sample type, instrument capabilities, etc.; regulatory requirements suchas FDA guidelines (e.g., the bioburden must be a measure of total viableorganisms as required by the FDA), review, expectations/acceptability;compliance requirements such as vendor audits, vendor support(instrument IOPQ or instrumentally observed perspectival quality),software validation, and documentation; and business requirements suchas industry trends, costs of implementation, cost per test, etc.

A few potential alternative methods for detection of microbialcontamination which are present within various embodiments of theinvention are listed in Table 3. Thus, for example, an alternative tocandling of eggs, and one embodiment of the current invention, comprisespre/post virus inoculation thermal imaging. In such embodiments, theinfrared radiation emitted by incubated eggs is captured with aninfrared camera. Using software, the captured images are converted intotemperature readings for the eggs. The camera is able to capturedifferences in temperature less than or equal to 0.01° C. Metabolicallyactive developing embryos lose heat slower than an infertile egg or adead embryo, thus, resulting in a higher temperature differential. Forexample, to set up, as an alternative to candling of eggs, thermalimaging of pre/post virus inoculation, a tray of eggs can be thermallyimaged (e.g., an infrared camera can be set below a tray-of eggs (e.g.,a tray with open-bottomed cells)). Software can then be set up tomeasure the bottom temperature of each egg (or side, top, etc.).Temperature decay rates of each individual egg can be evaluated, thus,allowing identification of the time to show maximum temperaturedifferential in problem eggs. Through such thermal imaging, temperaturedifferentials between live embryos and infertile and dead eggs can beidentified. See FIGS. 10 and 11.

In other embodiments herein, the current invention utilizes analternative to bioburden test on virus harvest, namely, MPN or MostProbable Number, which is based upon Bacteriological Analytical ManualOnline, January 2001, Appendix 2, Most Probable Number from SerialDilutions, FDA/CFSA-BAM. For example, an MPN Test can involve a 3replicate 96-well test, wherein 1:10 serial dilutions (e.g., 1:10,1:100, 1:1K, 1:10K, 1:100K, and 1:1000K dilutions) can be run intriplicate for 3 different samples on a 96 well microtiter plate withnegative controls. TSB can be added initially to all wells as a diluentand as an enriched media to the support the growth of microorganisms.Plates can be read visually or at 600 nm. MPN bioburden tests are quiteuseful in comparison to membrane filtration tests for detection ofcontamination. While membrane filtration tests can require (for 3samples) 15 TSA plates, a large sample volume, intensive amounts of timeand labor, can be difficult to automate and only sample at 1:10 and1:100 dilutions, a 96-well MPN test can (for 3 samples) only require one96-well microtiter plate (with controls), a small volume of sample, afew simple disposables and reagents, gives a dilution range from 1:10 to1: 100,000, and can also be visual read or automatably read with a96-well plate reader. The results of testing 70 samples usingconventional Bioburden and 96-well plate MPN were found in completeagreement with each other. Notably, for its intended purpose, the96-well plate MPN provided comparable results with a higher throughput.

As an alternative to the traditional compendial Mycoplasma test forVirus Harvest, the current invention, in some embodiments, comprises useof universal commercial standardized rapid nucleic acidamplification-based kits (e.g., PCR). The current compendial method(direct and indirect) detects all strains of contamination (includingavian M. synoviae and M. gallisepticum and human M. pneumoniae, i.e.,all avian and human mycobacterium strains). The alternative PCRdetection method comprises investigator-developed primer/probe sets forreal-time PCR that specifically detect a mycoplasma panel, and possiblygreater than 40 species based upon sequence homology of target gene(e.g., genus and/or species specific sequences on 16s and/or 23s rRNA)such as tubercle bacterial and non-tuberculous mycobacteria (e.g., M.abscessus and M. avium). Some embodiments herein utilize standardizednucleic acid amplification-based kits that rapidly detect tuberculebacteria and non-tuberculous mycobacteria, etc.

Group 2

Aspects of the current invention which fall into Group 2 include thosecorresponding to Step 4 through Step 6 in Table 1. After the process ofcorrect reassortment and cloning of reassortants (i.e., the 6:2viruses), such reassorted virus particles are further purified inembryonated hen eggs and the correct clones are expanded in quantity(again through growth in hen eggs) to generate a master virus strain(MVS) or master virus seed, which, in turn, is further expanded togenerate a master working virus strain (MWVS) or manufacturer's workingvirus seed. Many aspects of purification of virus particles from eggsand use of such purified virus to inoculate more eggs in order to expandthe quantity of virus particles are well known to those skilled in theart. Many such techniques are common in the current production of virusparticles and have been used for at least 40 years. See, e.g., Reimer,et al. Influenza virus purification with the zonal ultracentrifuge,Science 1966, 152:1379–81. For example, common purification protocolscan involve, e.g., ultracentrifugation in sucrose gradients (e.g.,10–40% sucrose), etc. Also, as noted herein, other procedures, etc.listed in other Groups are also optionally present within Group 2, e.g.,prevention of microbial contamination, etc.

Group 3

Aspects of the current invention which fall under the heading of Group 3include Step 7 through Step 11 in Table 1. These steps primarily dealwith the conditioning of the embryonated eggs (e.g., specific handlingand environmental conditions involved in the incubation of virusinfected eggs) and the harvesting and clarification of influenza virusfrom the allantoic fluid of the eggs.

For example, the current invention comprises conditioning, washing,candling, and incubating eggs which contain the reasserted virus to beused in a vaccine; inoculation, sealing, etc. of such eggs; candling ofsuch eggs; harvesting of the virus solution (e.g., the allantoic fluid)from the eggs; and clarification of the virus solution. Again, it shouldbe noted that several techniques applicable to the steps in Groups 2 areequally applicable to the steps in Group 3 (e.g., candling, etc.).Several aspects of the invention which comprise Groups 3 are well knownto those skilled in the art. Various aspects of candling of eggs invirus production, as well as inoculation of eggs with viruses andwashing, incubating, etc. of such eggs are well known techniques in theproduction of virus/vaccines in eggs. Of course, it will be appreciatedthat such well-known techniques are used in conjunction with the uniqueand innovate aspects of the current invention.

Rocking

One drawback in culturing some types of influenza strains (e.g.,especially influenza B strains such as Victoria/504/2000) is that theydo not produce as high a titer as other strains when grown in eggs. Forexample, if a first strain (e.g., an influenza A strain) produces atiter of 10⁸ or 10⁹ log (i.e., 10⁸ or 10⁹ virus particles permilliliter) and a second strain (e.g., an influenza B strain) onlyproduces 10⁷ virus particles per milliliter, then the second strain mustbe, e.g., grown in a greater quantity of eggs, or the first strain mustbe held until the second strain is grown in a second production, etc.

Thus, one aspect of the current invention is to rock or gently agitatethe eggs in which the virus strains are incubated (i.e., after the eggsare inoculated with the virus). It should be noted that the exactmechanism used to achieve such rocking is not limiting. For example, theeggs are optionally rocked on a shaking platform or rocking platform(e.g., as is used to incubate bacterial culture flasks, as is used inegg incubators, etc.). In some embodiments, the eggs are rocked fromabout 1 cycle per minute or less to about 2 cycles per minute or more.In this context, “cycle” should be taken to mean the traveling of theeggs through a full range of motion. In yet other embodiments, the eggsare rocked from abut 0.5 cycles per minute or less to about 5 cycles perminute or more. In some embodiments, the eggs are rocked at about 1cycle per minute. When rocking was added to the incubation steps inGroup 3 (i.e., post inoculation) the titer of a B-Victoria influenzastrain increased by 0.4 log over a control group of eggs which was notrocked.

Filtering and Warming

Yet another aspect of the invention that falls under Group 3 involvesthe effect of viral allantoic fluid (VAF) temperature on virus potencylosses during sterile filtration (typically through 0.2 um filters). Invarious embodiments of the current invention, virus particles areharvested from allantoic fluid and then put through a process involvingwarming of the fluid followed by filtration of the fluid. See, e.g.,Steps 10 and 11 in Table 1. Such steps are desirable for severalreasons. For example, as pointed out herein, presence of allantoic fluidand debris in vaccine preparations can lead to allergic reactions. Also,quite importantly, filtration removes bioburden (bacteria) from thesolutions. All VH (virus harvest) solutions containing bioburden must bediscarded. This is also true in intranasal application oflive-attenuated virus vaccines. Thus, the aspects of the currentinvention which allow filtration and clarification of live attenuatedvirus in order to remove and/or reduce the presence of such bioburden,etc. is quite desirable.

The effects of viral allantoic fluid (VAF) temperature and warming timenecessary to filter a cold-adapted (ca) virus strain (e.g.,A/Sydney/05/97, H3N2 type) with acceptable potency loss throughsterilizing-grade filters is used as an example herein. Conditions toacceptably filter A/Sydney/05/97 are discussed, as well as the resultsof five additional cold adapted influenza strains (namely: 2×H1N1,1×H3N2, 2×B) being filtered under similar conditions.

Three independent assays (TCID₅₀, neuraminidase, and hemagglutinin) wereused to characterize viral allantoic fluid throughout the filtrationprocess. The data demonstrate that the addition of a warming step (e.g.,exposure to the temperature of 31±3° C. up to 60 minutes prior tofiltration) to the filtration process reduced the potency losses toacceptable levels (0–0.3 log₁₀ TCID₅₀) compared to the sterilizing-gradefiltration performed without warming step for A/Sydney/05/97. In otherembodiments, the warming temperature is optionally over 28° C., or from28 to 36° C. for a period of time of at least 30 minutes, or, in otherembodiments of from about 60 to 240 minutes. It will be appreciated thatthe warming process can, indeed, continue for long periods of time, butthat after greater lengths of time, the loss in potency due to virusstability loss at such elevated temperatures becomes measurable anddetrimental. The added warming step did not contribute to additionalpotency losses for other tested strains for the times tested, indicatingthe warming step is an acceptable process step for sterilizing-gradefiltration of cold-adapted influenza viruses (CAIV).

As described herein, the current FluMist™ manufacturing process usesembryonated chicken eggs to generate master virus seeds (MVS),manufacturer's working virus seeds (MWVS) and virus harvests (VH). SeeStep 6 in Table 1. The seeds and viral harvest may contain bioburden(typically bacterial contamination), which would cause the seed or bulkvirus product lots to be rejected in the vaccine production process.Through previous studies to evaluate the use of filtration for viruscontaining allantoic fluids, indication had been that bioburden can bereduced by the introduction of a filtration step in the process.However, based on previous work, such filtration is problematic withparticular viral strains (e.g., A/Sydney/05/97). Based on such studies,design proposals have been made for filtration rigs comprised of asterile plastic media bag connected to a pre-filter and 0.2 millimetersterilizing-grade filter combination with various associated filling,dispensing and sampling lines (see below). Of course, it will beappreciated that specific listing or description of particular producttypes used, sizes, etc., is not to be considered limiting on the currentinvention unless specifically stated to be so.

As seen in such studies, the majority of tested cold-adapted (ca) viralstrains can be filtered with minimal potency loss though a SartoriusSartoclean CA pre-filter followed by a Sartorius Sartopore 2 as thesterilizing-grade filter. However, other filtration studies withA/Sydney/05/97 resulted in potency losses of between 0.7 to 1.4 log₁₀TCID₅₀/mL. Further studies revealed that this loss occurred across theSartorius Sartopore 2 sterilizing-grade filter. Again, it should benoted that other filter brands and/or filter types are optionally usedin such steps and that recitation of particular filter names/typesshould not be construed at limiting.

The purpose of the first set of experiments shown below was to test theeffect of VAF temperature on virus potency loss during filtration. Thesecond part of the study was designed to define the appropriate warmingtime of VAF prior to filtration. The cold-adapted (ca) A/Sydney/05/97virus strain (H3N2 type) was used as a model strain to determine thewarm-up conditions because, as stated previously, large potency losseshave been observed during filtration of this strain.

The third part of this example evaluated the effect of warming the viralallantoic fluid (VAF) on potency losses caused by filtration for severalother monovalent virus strains. Five CAIV strains (2×H1N1, 1×H3N2 and2×B) were used in these runs. All experiments were performed at the CAIVseed-scale (MVS and MWVS) using 1.0–3.0 L of sucrose phosphate glutamate(SPG) stabilized VAF and appropriately scaled filters, i.e.approximately 1:30 to 1:10 of proposed maximum VH process scale, priorto removal of testing samples. Typical process scale is up to about 33 Lof stabilized VH per filtration rig. Such volume typically works wellwith 50 L bags chosen for filtration rigs and has a reasonable safetymargin for volume that can be filtered using standard 10″ filtercapsules. However, such volume is often too large fordevelopment/exemplary work; thus, a 1/10^(th) scale filtration wasperformed (i.e., about 3 L).

Virus propagation for such temperature/filtration steps can be performedaccording to commonly known methods in the art, and/or using otheraspects of the current invention (see, above and below) usingcold-adapted (ca) influenza strains summarized in Table 4.

Samples from all stages of the experiment were assayed for potency bymeasuring the Tissue Culture Infectious Dose (TCID₅₀) in a manual assay(see below for other aspects of TCID₅₀ measurements). Neuraminidaseactivity (NA) and Hemagglutinin activity (HA) were also measured.

A series of filtrations through Sartorius Sartoclean CA/SartoriusSartopore 2 filter combinations were performed in order to evaluate theeffect of VAF temperature prior to filtration on: potency (TCID₅₀/mL),neuraminidase (NA) and hemagglutinin (HA) activity losses.

During the virus harvest, VAF was pooled into 1 L PETG bottles. Once therequired volume of unstabilized VAF was collected and pooled, thefiltrations were performed. The temperature (start-up temperature) ofthe unstabilized VAF at this stage was 15±3° C. The total warming timewas defined as the time the VAF was in the 33±1° C. water bath andconsisted of the warm-up time (from 15±3° C. to 28±3° C.) and warm-holdtime (time greater than 28° C., e.g., at a set point).

The Part 1 VAF temperature effect studies (see below) were performedwith the cold-adapted (ca) A/Sydney/05/97 virus strain (H3N2 type). Part2 of the example focused on determination of the optional warm-hold time(“time at the temperature”). In part 3, the effect of the previouslydetermined warm-hold time on five other strains (Table 5) was tested. Inall parts of the example, 1.0–3.0 L of sucrose phosphate glutamate (SPG)stabilized VAF, typical virus seed-scale, and approximately 1:30–1:10 ofproposed mVH process scale, were filtered through the rigs.

In the current typical manufacturing processes, after harvest, VH iscentrifuged, stabilized and frozen for further transportation. In theseexamples, a sample of VAF withdrawn from the un-stabilized pool wascentrifuged and stabilized with SPG, similarly to current manufacturingprocesses and this served as a control for filtered VAF in all parts ofthe current example.

Part 1: Temperature Effect on A/Sydney/05/97 Virus Titer Changes DuringFiltration

In order to determine the effect of temperature on potency loss, twosets of filtration experiments at various temperatures were performed.Each set consisted of three parallel experiments performed on the sameday with VAF collected from the same batch of eggs. In theseexperiments, after harvest, VAF was stabilized with SPG, split intothree pools and exposed for 60 minutes prior to filtration to either5±3° C. (refrigerator), 20±3° C. (bench top) or 31±3° C. (water bath).During this time, VAF in the bottle was mixed by inverting every 10minutes. After the temperature treatment, it was filtered throughSartoclean CA and Sartopore 2 filters. In the control experiment, VAFwas centrifuged and stabilized. TCID₅₀ results of filtration underdifferent conditions were compared to each other and the control.

To determine the effect of VAF temperature on potency loss, VAF wasexposed for 60 minutes prior to filtration to 5±3° C., 20±3° C or 31±3°C. The potency change, neuraminidase and hemagglutinin activitydifference between centrifuged stabilized and post-filtration materialwith different temperate treatment is summarized in Tables 5–10. As canbe seen, filtration of cold (5±3° C.) and room temperature (20±3° C.)VAF resulted in potency losses between 0.7 and 1.0 log₁₀ TCID₅₀/mL (see,Tables 5 and 8). However, there was no post filtration titer loss(compared to the centrifuged stabilized VAF) when VAF was warmed up to31±3° C. for 60 minutes (30 minutes warm up time +30 minutes warm-holdtime at the set point temperature). See, Tables 5 and 8. Additionally,the post filtration neuraminidase activity levels were higher in thefiltration performed after VAF was warmed up to 31±3° C. compared to thelevels observed in cold and room temperature filtrations. See, Tables 6and 9. Addition of the warm-up step also reduced hemagglutinin activitylosses. See, Tables 7 and 10.

Part 2: Determination of the Warming Time Required for AcceptableFiltration Potency Losses of A/Sydney/05/97

In order to determine the necessary warming time, a series ofexperiments were conducted with VAF warmed to 31±3° C. prior tofiltration in a water bath. In a control experiment, VAF was filteredimmediately after stabilization with SPG. In all experiments, warmingtime was defined as the total time (warm up time plus warm-hold time)VAF was in the water bath (i.e., at 31±3° C.). VAF in the bottle wasmixed by inverting every 10 minutes. After temperature treatment it wasfiltered through Sartoclean CA and Sartopore 2 filters. In the controlexperiment, representing the current manufacturing process, VAF wascentrifuged and stabilized. TCID₅₀ results of filtration under differentconditions were compared to each other and the control.

To determine the warming time prior to filtration that is required tofilter ca A/Sydney/05/97 a series of experiments was conducted whereinVAF was warmed to 31±3° C. prior to filtration for 30, 90 or 180 minutesin one set of experiments and 30, 60 or 90 minutes in another set ofexperiments. In the control experiments, VAF was filtered withoutwarming immediately after stabilization with SPG. The virus potency,neuraminidase and hemagglutinin levels between filtered VAF and controlare summarized in Tables 11–16.

The data demonstrate that the exposure of VAF to 31±3° C. reduced postfiltration virus potency losses and allowed partial recovery ofneuraminidase and hemagglutinin activities. See, Tables 11–13. Thetemperature of un-stabilized VAF at the beginning of the experiments(post harvesting and prior to warming) was 15±2° C. The warm up timerequired for 1–1.5 L of VAF to reach 31±3° C. was about 20–30 minutes.Thus, a 30-minute total VAF warming time results in 0–10 minutes VAFwarm hold time at 31±3° C.

The minimum warming time required to minimize filtration potency losseswas determined in a second series of experiments. See, Tables 14–16(first set) and Tables 17–19 (repeat set). The post filtration potency,HA and NA losses were observed in 0 and 30 minutes total warming timeexperiments. In 60 and 90 minute total warming time (warm-hold of 30–40and 60–70 minutes at 31±3° C.) experiments, post filtration viruspotency and HA and NA levels were similar to the control (centrifugedstabilized VAF) samples. See Tables 14–19.

Part 3: Effect of Warming on Other Strains

A series of experiments was conducted with 5 strains other thanA/Sydney/05/97, i.e., 2×H1N1, 1×H3N2, and 2×B, in order to assess theeffect of the warm up step on filtration of influenza virus strainsother than A/Sydney/05/97. Each strain was tested twice. VAF was warmedto 31±3° C. for 60 minutes (30 minutes ramp up time+30 minutes time atthe temperature) prior to filtration. After temperature treatment, itwas filtered through Sartoclean CA and Sartopore 2 filters. In a controlexperiment, VAF was filtered immediately after stabilization with SPG atroom temperature. TCID₅₀ results of filtration under differentconditions were compared to each other and control experiments.

For the additional 5 cold-adapted influenza virus strains tested, ashort exposure (total warming time of 60 minutes) to 31±3° C. (warm-holdtime of 30–40 minutes at set point temperature) contributed to thereduction of post filtration potency losses compared to the experimentswithout temperature treatment for A/Sydney/05/97 and B/Victoria/504/2000and did not impact potency for the other strains. The potency(TCID₅₀/mL), neuraminidase and hemagglutinin levels from theseexperiments are summarized in Tables 20–25, below.

As can be seen from the tables, the aspect of the current inventioncomprising warming to 31±3° C. or optionally even up to 36° C.(warm-hold time of 60 to 90 minutes for 1–1.5 L of VAF in bottles) ofthe stabilized viral harvest prior to filtration through Sartoclean CApre filters and Sartopore 2 sterilizing grade filters resulted inacceptable reduction of virus potency (0–0.3 log₁₀ TCID₅₀/ml) forA/Sydney/05/97. In the control experiments, when A/Sydney/05/97stabilized viral harvest was filtered without warming, titer losses wereup to 1.0 log₁₀ TCID₅₀/ml.

As is also seen from such tables, for all 6 cold-adapted influenza virusstrains tested, a short exposure (warm up and warm hold time of 60minutes) at 31±3° C. (warm-hold of 30–40 minutes at 31±3° C.) eitherdecreased the potency losses or did not contribute to additional potencylosses during filtration. In all experiments, the post filtration titerloss was not higher than 0.3 log TCID₅₀/ml. The reduced activity lossesof the viral surface proteins (neuraminidase and hemagglutinin) ofwarmed filtered VAF compared to not warmed, support the decreasedpotency loss data shown by TCID₅₀ assay.

Thus, the data verifies that some embodiments of the current inventionwhich comprise a warming time required to filter CAIV (MVS, MWVS or VH)have acceptable potency losses of 60 minutes (time to warm up the VAF to31±3° C. and warm hold (time at the set point temperature) for at least30 minutes). Such warming tolerance is a novel and unexpected result,especially in light of other filtration attempts. See above. Again, aswill be appreciated, the embodiments of the current invention comprisingheating/filtration steps are not limited by the above examples. In otherwords, e.g., other filters and filter types, etc are optionally used,without deviating from the invention.

Group 4

Group 4 of the aspects of the current invention comprises, e.g., Steps12–15 of Table 1. Such steps primarily concern stabilization (e.g.,through addition of components, alterations in buffer/NAF ratios, etc.)and assays of potency/sterility of virus containing solutions. In someembodiments, the final viral solutions/vaccines comprising live virusesare stable in liquid form at 4° C. for a period of time sufficient toallow storage “in the field” (e.g., on sale and commercialization whenrefrigerated at 4° C., etc.) throughout an influenza vaccination season(e.g., typically from about September through March in the northernhemisphere). Thus, the virus/vaccine compositions are desired to retaintheir potency or to lose their potency at an acceptable rate over thestorage period. For example, if a 0.3 log potency loss were acceptableand the storage period were 9 months, then an 0.05 log/month decrease inpotency would be acceptable. Furthermore, use of FFA allows a greaterlatitude in terms of acceptable loss. For example, if a loss of up to0.75 log were allowed, a rate of less than or equal to 0.09 log/monthwould be sufficient to allow stability of materials stored continuouslyat refrigerator temperature (e.g., 4° C.). In other embodiments, suchsolutions/vaccines are stable in liquid form at from about 2° C. toabout 8° C. In yet other embodiments, the solutions/vaccines are stableat room temperature. Typical embodiments herein do not exhibit adecrease (or exhibit small decreases) in immunogenicity due to the NAFdilutions (see below).

Concentration/Diafiltration of Virus Harvests

In some embodiments herein, virus harvests are optionally concentratedusing a appropriate column. Influenza virus solutions can beconcentrated without loosing appreciable viral potency/activity. Suchconcentration without loss of potency is a quite surprising resultbecause previous literature, etc. showed a loss of virus activity withconcentration. Viral concentration can be done at a number of points inthe purification/production process, e.g., as illustrated in Table 1, inorder to enhance the viral particles and remove other proteins, RNA,etc. For example, concentration can be done prior to potency assaying,or even after potency assaying, etc., but in many embodiments is donewithin/amongst the steps categorized in Group 4. Concentration of virusparticles can be useful for purification, vaccine preparation, and foranalytical characterization. See, e.g., Methods and Techniques inVirology, Pierre Payment and Michel Trudel, Marcel Dekker, Inc., (1993).Due to the low amount of virus in some VAF samples, the direct analysisof the virus particles precludes some of the analytical techniques likeAnalytical Ultra Centrifugation (AUC), Disc Centrifuge, Matrix AssistedLaser Desertion Ionization (MALDI), and particle counting.

Prior traditional viral concentrations from egg NAF, etc. were done viagradient purification centrifugation. See, e.g., Concentration andPurification of Influenza Virus from Allantoic Fluid, Arora et al.,Analytical Biochemistry, 144:189–192 (1985). Embodiments herein,however, utilize size exclusion columns. Concentration can be usedwhether the virus is produced via egg production, cell cultureproduction (e.g., Vero cells), plasmid rescue production, etc. Also, theconcentration steps can be performed on a number of different virusesand/or virus strains (e.g., both influenza A and influenza B strains areamenable to such actions) as well as between different lots of onestrain, e.g., to ensure product quality. Additionally, size exclusioncolumn concentration can often be used as a track on the amount of virusparticles within a harvest, e.g., within an egg, etc. Thus, for example,a peak area (i.e., of virus eluted from the column) can be used insteadof, or in addition to, TCID₅₀ measurement of such solutions. Suchtracking is especially useful for virus produced in eggs. Additionally,concentrated and purified virus material can optionally be a startingmaterial for generating pure HA, NA and other viral components forfurther studies. Furthermore, SEC purified virus can provide a betterinsight into the virus structure and the binding mechanism with the hostcells. Because in most of the VAF (virus/viral allantoic fluid)materials, virus particles are below the detection limit of UV, theconcentration of the virus particles is quite helpful for furthercharacterization.

In concentration of virus harvest, a size exclusion column, e.g., MidGeeor QuixStand (Amersham) with hollow fiber filter under pressure can beused to remove impurities and/or unwanted buffers/fluids. Theconcentrated virus is, thus, also more easily suspended or stored inspecific buffers/stabilizers. See below.

To illustrate the concentration of a virus harvest sample, an influenzaharvest of A/New Caledonia was concentrated and analyzed from VAF bycross flow filtration. Of course, again, it is to be emphasized that thetechniques, etc. of this section are not be limited to particularstrains/types of viruses. Such concentration concentrated the virusparticles, removed a majority of impurities and retained virusinfectivity. As illustrated, the virus infectivity was checked by CELISA(TCID₅₀). Hemeagglutination by HA assay, neuraminidase activity, SECanalysis, NAF by RHPLC, and RNA by RTPCR were also done.

The virus concentration in the example below was achieved by usingAmersham's Cross Flow Filtration Unit MidGee. MidGee is capable ofconcentrating 100 or 200 ml to 10 ml in 2–3 hours. Similarly, QuixStandcan be used for concentrating the virus particles from 2 liters to 100ml in 4 to 6 hours. Concentration of virus not only enhances the virusparticle count, but also removes a majority of other impurities like eggproteins, RNA, and small molecules like uric acid.

The virus used in the following example was A/New Caledonia/20/99. NAFcomprised cold adapted influenza virus. Chicken blood was from ColoradoSerum Company (Denver, Colo.). The instrument used for concentration wasfrom Amersham Biosciences (A/G Technology Corporation), and was a MidJetSystem with Peristaltic Pump (Watson Marlowe). The column used forconcentration was from Amersham Biosciences (A/G Technology Corporation)and was a MidGee Hoop Cross Flow Filter with a nominal molecular weightcut-off of 750,000. Yet again, however, it is to be emphasized that useor recitation of particular models, producers, etc. of equipment are notto be construed as limiting upon the current invention. The buffer usedfor washing in this example was 1×-SPG.

For SEC, the instrument used was a Heweltt Packard HP 1100 HPLC systemwhile the column was an Ultrahydrogel 1000 from Waters with a size of7.8×300 mm. The buffer with the SEC was Dulbecco's Phosphate BufferedSaline from Hyclone Solvent. For the SEC, the method comprised anisocratic condition with a flow rate of 0.5 ml/min, monitored at 210 and280 nm. For the RHPLC, the instrument was from Waters and the column wasa YMC C4 (reverse phase), 2.1×250 mm, 5 um, 300 A. The method for theRHPLC was: Mobile Phase—A: 0.1% TFA in water, B: 95% CAN 0.09% TFA;Elution Conditions—Variable gradient, 13–100% B ; Flow Rate: 0.2 ml/min;Column Temp, −45 C; Injection Volume—50 ul; and Detection—214 nm.

As shown in FIG. 12, Step 1, 150 ml of A/New Caledonia/20/99 wasconcentrated by a MidJet instrument in a cold room. The pressure betweenthe inlet and outlet was maintained between 5 to 10 PSI. Aftercirculating through the cross filter for two hours, 150 ml of the 1×sample was reduced to 15 ml of 10× concentrated sample (Step 2). Thepermeate was collected separately and stored for further analysis. Foranalytical characterization, 4 ml of the 10× sample was removed (Step3). The remaining 11 ml of the 10× sample was diluted to 110 ml with1×-SPG, and was further concentrated down to 11 ml by removing the1×-SPG as a permeate. The permeate carries most of the impurities fromthe retentate. This step was repeated five times with 1×-SPG as shown inStep 4 and Step 5. The washed permeate was saved for further analysis.The first and second wash showed yellow coloration. This is thought tobe due to the removal of egg proteins and other small moleculeimpurities. The yellow color in the permeate disappeared after the 3rdand 4th wash. Following the 5th wash, the sample was diluted with 1×-SPGto 110 ml to bring the concentration back to 1×. At step 6, 10 ml of the1×-W was reserved for the assay. The remaining 100 ml of the 1×-W wasfurther concentrated down to 10×-W (Step 7). This concentrated samplewas aliquoted into 1 ml quantities for further analysis.

All the samples were analyzed by SEC chromatography. The Ultrahydrogel100 column was used for the analysis with DPBS as a solvent. Even thoughthe data was collected at 220, 260 and 280 nm, for discussion purpose,the comparison was done with the 220 nm peak areas. The chromatogrampeaks were classified into three major groups: one for virus (retentiontime around 10.6 min), one for impurities group-1(retention time 18 to21 min), and one for impurities group-2 (retention time 21 to 27 min).Three NAF proteins Ovalbumin, Conalbumin and Ovomucoid elute aroundretention time 18–21 min. See FIG. 13. Lysozyme elutes around 27.0 min.It is thought that Group-2 impurities consist of small molecules such asuric acid and other uncharacterized materials. All the washes werechecked by analytical SEC chromatogram under identical condition as thevirus analysis. The CELISA, HA assay, NA assay, and RTPCR were carriedout by different groups.

SEC Analysis and CELISA

The neat sample, 1× showed the virus peak at 11.1 minutes with a peakarea 1,221. See FIG. 14. However, the concentrated 10× sample showed apeak area 11,192, see, FIG. 15, and the increment in the peak are wasabout 9.16 times compared to 1×. See Table 26, 11,192/1221. This isbased on the previous experiments showing linearity between the peakarea and the amount of virus sample injected. During the concentration,without any washes, some impurities have been removed but notsignificantly. See Table 26, FIG. 16 a–b. The impurities group-1 andgroup-2 showed increment in the peak size between 1× and 10× (Table 26).Correspondingly the TCID₅₀ was increased from log 9.1 to log 10.0 (Table27). During this step, 95.9% of the infectivity was retained. This dataindicates that concentrating the 1× sample to 10× sample retained theinfectivity quite well.

After the 5th wash with 1×-SPG, the virus peak area of the sample 1×-W,retained as 1005 compared to 1221 before the wash (Table 26). Recoveryby peak area between 1× and 1×-W was about 82% (1005/1221). By comparingthe 1× and 1×-W chromatogram (see FIG. 17), it shows that impuritiesgroup-1 and group-2 were significantly reduced (Table 26). The 1×-Wshowed a small decrease TCID₅₀ value (Table 27, 1×: 9.1, 1×-W: log 8.9).The recovery of infectivity was about 98.99% between 1× and 1×-W (log8.9/log 9.1). The washing step improved the quality of the virusmaterial by removing NAF proteins and other components.

Similarly, by comparing the 10× and 10×-W, the impurities group-1 andgroup-2 was removed to a great extent (Table 26, FIG. 18). By goingthrough the 5 washes, the virus peak area of 10×: 11,192 was reduced to10×-W: 10, 282 (Table 26, 91.86% by peak area). The TCID₅₀ was changedfrom log 10.0 (10×) to log 9.9(10×-W) with the recovery of 99.56% (Table27).

By comparing the 1×-W and 10×-W chromatogram, the peak area increased by10 times. See Table 26, 1×-W: Peak Area: 1005 and 10×-W: 10,282. TheTCID₅₀ value also increased one log (Table 27, 1×-W: log 8.9 and 10×-W:log 9.9). Since, the 10×-W was concentrated from 1×-W in one step, noloss in either activity or in the peak area was seen (10×-W: Peak area10282 and 1×-W Peak Area 1005).

The permeate showed virus peak at 10.4 min with the peak area 25. Thiscould be due to the loss of a very small amount of virus particles orsome other proteins eluting along with the virus in 1× sample. Most ofthe impurities were eluting in group-1 and group-2. See Table 26. TheCELISA values showed the infectivity was below the limitation ofdetection. This shows that there was not many virus particles elutingthrough the membrane during the concentration procedure.

The five washes improved the quality of the virus by removing most ofthe impurities of group-1 and group-2. This is illustrated in the Table26 and FIG. 19. Group-1 and group-2 impurities were significantlyremoved after the 2nd wash. After the 5th wash the curves reached aplateau. Even after the 5th wash, the samples 1×-W and 10×-W showedimpurities group-i and group-2 in a very low amount. See FIG. 20. Theidentity of the peak at 19.208 min was confirmed as ovalbumin byisolating from the 10×-W sample. SDS-PAGE also confirmed the result.

HA Assay

The sample 1× and 1×-W showed HAU 1024. See FIG. 21. The concentrated,but not washed, 10× showed at HAU 8192. However, 10×-W showed a falsenegative at HAU 2 and 4. This may be due to the large amount of viruscompared to the chicken RBC. High amounts of neuraminidase reverse thehemeagglutination process. See, Virus cultivation, Detection, andGenetics, S. J. Flint, L. W. Enquist, R. M. Krug, V. R. Racaniello andA. M. Skalka, “Principles of Virology,” ASM Press, Washington, p 34,(2000). The absence of HAU in the permeate shows that there was not muchvirus eluting in the step 1. See FIG. 12.

NA Assay

The neuraminidase assay illustrated that 10× diluted back to 1× showssome decrease in activity in comparison with 1×. See FIG. 22. This wasthought to be due to the loss of free NA protein from the VAF material.This was supported by a small amount of NA in the permeate. Samples 1×-Wand 10×-W diluted back to 1×-W retained the activity at the same level.This was because the sample 10×-W was concentrated directly from 1×-W.All the washes have the activity below the detection level.

RHPLC

Egg protein analysis had been optimized previously by RHPLC, therefore,all the present materials in the example were analyzed under identicalcondition, e.g., C4 column with 0.1% TFA/Acetonitrile gradient andmonitored by 214 nm. The elution pattern of the ovomucoid, lysozyme,conalbumin and ovalbumin is shown in FIG. 23. The 10-× sample, beforeany wash, showed all the egg proteins. This matches the retention timeof the control sample. Also 10× showed unidentified viral protein peakslabeled as U1, U2 and U3. Completely washed samples 10×-W and 1×-Wretained the viral proteins U1, U2 and U3. The 10× and 10×-W samplescontained the same amount of U1, U2 and U3 proteins. Because the ratioof these proteins was the same, the proteins might be generated from thevirus particles during the exposure to acetonitrile. However, theovomucoid, lysozyme and conalbumin have been completely removed from 10×by washing with 1×-SPG for five times. Notably, in contrast, the mostobvious protein peak is ovalbumin, which is still eluting along with10×-W and 1×-W samples. Even though 10×-W and 1×-W have gone 6 and 5washes, still ovalbumin bound to the virus. This may be due to thestrong interaction between HA proteins and ovalbumin. This data alsopresented in the bar graph form as in FIG. 24.

The permeate and all the washes were checked by RHPLC. See FIG. 25. Thepermeate contains all the NAF proteins and other unidentified peaks.Ovomucoid was removed by two washes (see FIG. 26); lysozyme by 2 washes(see FIG. 27); conalbumin by two washes (see FIG. 28); and ovalbumin wasdepleted gradually, but about 5% remained even after wash number. 6. SeeFIG. 29.

Agilent Bioanalyzer

Simultaneously, ovalbumin was estimated by Agilent Bioanalyzer as shownin FIG. 30. Just by the concentration, without any washing step,ovalbumin was considerably removed from 1× to 10×. The first permeatecarried most of the ovalbumin. RHPLC showed ovalbumin in all the washes,but in the Bioanalyzer analysis it reached below the detection limit.The 10×-W sample was diluted ten times to reach the concentration closeto 1×-W sample, and it showed a small amount of ovalbumin. Based on thisdata, 95% of the egg proteins were removed by the concentration andwashing steps.

SDS-PAGE and Western Blot

In comparison to 1× (lane 2), 10× (lane 9) contains more intensemultiple silver stain bands. See FIG. 31. 10×-W (lane 10) showed fewernumber of bands compared to 10×. This was due to the removal of NAFproteins and other impurities. Similarly 1×-W (lane 8) appears cleanerthan 1×. Samples 1×, 10× diluted to 1× (3rd Lane), and 10×W diluted to1×-W (4th lane) contain the same quantity of virus except differentdegrees of improvement in the removal of impurities. Obviously, 10×-Wdiluted to 1×-W shows clearer viral protein bands. However, this samplestill contained an ovalbumin band. This is compared with NAF proteins inlane 6. The 10×-W sample was further purified by analytical SEC columnand the fractions were collected. See FIG. 32. The fraction collected at19.1 min was checked by SDS-PAGE, and this fraction contains mostlyovalbumin protein (lane 5). This lit up in the Western Blot againstanti-NAF. This is additional evidence to show that ovalbumin stronglybinds to the virus even after 6 washes. The anti-NAF gel was strippedand probed with chicken anti-A/New Caledonia. Distinct bands wereobserved representing the viral proteins, HA₀ and HA₂ or M protein. SeeFIG. 31.

RTPCR

RTPCR showed that the RNA was about a log higher between 1× and 10×. SeeFIG. 33. Similarly, there was about a ten-fold increase in the viral RNAbetween 1×-W and 10×-W. This indicated that most of the virus wasretained during the concentrations step. Permeate does not have anydetectable viral RNA, but the 1×-SPG washes showed a very small amountof RNA. This may be due to small amount of virus undergoing shearingduring the circulation or some viral RNA bound to the filtrate andreleased latter slowly during the washing cycles.

In summary, the concentration of the A/New Caledonia/20/99 was achievedby using a cross-flow-filtration device. The infectivity of the virusparticles was retained during this procedure, and it was confirmed byCELISA assay. Washing the concentrated material by 1×-SPG improved thequality of the virus by removing other impurities. Even after the 5thwash a small amount of ovalbumin was strongly bound to the virus. Thismay be due to the strong interaction between ovalbumin and the HA or NAprotein. RHPLC and SDS-PAGE and Western Blot support thisprotein-protein view. The increase in the quantity of RNA between theneat and concentrated sample indicates that the majority of the virus isrecovered by this procedure.

Similar techniques are also applicable for use with virus samples fromcell culture, e.g., influenza samples grown in cells such as Vero cells,etc. To illustrate such, three viruses were grown in Vero cell culture,namely, A/Beijing (A/H1N1) used as is; A/Panama (A/H3N2) concentratedfrom 2L to 100 ml or 20×; and B/Hong Kong concentrated from 2 L to 10 mlor 200×. It will be appreciated that since virus yields from Vero cellsare typically lower, the embodiments of the current section canoptionally be used to concentrate the virus samples. Similar to theabove illustration an Amersham MidGee and a QuixStand Instrument wereused for the virus concentration

FIGS. 34–35 show monitoring of A/Beijing cell culture propagation by SEC(FIG. 34) and A/Beijing Vero cell culture harvest (FIG. 35). As can beseen SEC is an efficient technique for monitoring the virus propagationin a short time. The amount required for such monitoring is alsotypically small (e.g., 100 ul). FIG. 36 illustrates concentration of a 2liter sample of an A/Panama cell culture sample. Two liters of virusharvest were concentrated down to 100 ml by QuixStand. See above. TheTCID₅₀ of the 1× mixture was non detectable, but the TCID₅₀ of the 20×mixture was 4.4. There was a peak area ratio of 20× to 1×. Theconcentration of the Panama cell culture sample illustrates theadvantages of cross-flow filtration, e.g., virus particles can beefficiently enhanced, low molecular weight impurities can be removedfrom the solution and diafiltration can be done for further “clean-up”of the solution. FIG. 37 shows concentration of 2 liters down to 10 mlof a Vero cell grown culture of B/Hong Kong. At 1× the log10 TCID₅0/mlwas 4.7, while at 18.8× it was 5.8 (the theoretical for such being 5.95)and at 200× it was 6.95 (the theoretical of which being 7.00).

From the above figures it can be seen that SEC is a useful technique formonitoring virus growth in cell culture samples; very low titer viruscan be assayed after concentration of virus samples; and low titer viruscan be assayed after concentration.

Stabilizers/Buffers

The invention comprises compositions of virus solution and methods ofcreating the same. Such compositions optionally comprise variousdilutions of NAF (typically unfractionated NAF) comprising the virus ofinterest and combinations of, e.g., sucrose, arginine, gelatin, EDTA,etc. as detailed herein. As will be noted, various compositions hereincomprise from 10% to 60% NAF. NAF can possibly contain various enzymessuch as nucleases lysozymes, etc. which could adversely affect thestability of virus compositions. Such methods and compositions arepreferably stable (i.e., do not show unacceptable losses in potency)over selected time periods (typically at least 6 months, at least 9months, at least 12 months, at least 15 months, at least 18 months, atleast 24 months, etc.) at desired temperatures (e.g., typically 4° C.,5° C., 8° C., from about 2° C. to about 8° C. or greater than 2° C.,etc.). Preferred embodiments show no decrease in potency over thedesired storage period. Other embodiments show less than 10% decrease,less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%decrease. The potency of virus compositions herein was measured in FFUor fluorescent focus units (see below in description of FFA assay). Atarget FFU value is typically set based upon the virus concentration ata time zero (e.g., due to dilution of the NAF, etc.). Preferredembodiments, thus, show little or no decrease from the starting value.In various compositions herein, the virus solutions comprise from about5% to about 10% sucrose, from about 1% to about 4% arginine, and fromabout 1% to about 4% gelatin. Some preferred embodiments comprise about7–10% sucrose, about 2% arginine, and about 2% gelatin. In someembodiments, the stability is measured after storage of the virusformulation at the desired temperature in a FluMist®applicator/accuspray device or other similar device.

In some embodiments, the invention comprises compositions comprisingstabilizer of, e.g., arginine (of pH from about 7.0 to about 7.2),either in combination with, or in place of gelatin or gelatin relatedand/or derived products (e.g., gelatin hydrosylate). See, Steps 12 and15 in Table 1. However, current regulatory concerns regarding thepotential of unintentional contamination from animal and animal-derivedproducts such as gelatin, collagen, etc. (e.g., by such problems asprions, mycoplasma, or host-derived viruses), as well as concernsregarding the potential for allergenicity of animal derived products,has lead to the need for non-animal based stabilizers. Arginine usedeither alone or in combination with additional excipients such as metalion chelators (e.g. ethylenediaminetetraacetic (EDTA) and/or its salts)or other amino acids (e.g. Histidine and/or its salts) offers thepotential of stabilization of cold-adapted influenza virus preparationswith a non-animal derived excipient.

In various embodiments, the arginine optionally comprises either a saltwith an inorganic acid or a salt with an organic acid. Of course, thesalt typically comprises a pharmaceutically acceptable salt since it isto be used as a vaccine component. Typical preferable salts comprise,e.g., hydrochloride, citrate, and sulfate. The amount of suchstabilizing agent used is not limited to specific particular ranges,however, typical amounts used range from about 5 mg to about 60 mg per 1mL of the virus solution. The amount used may preferably be from about10 mg to about 50 mg, and more preferably, from about 10 mg to about 25mg per 1 mL of the virus solution. In other embodiments, the amount usedmay range from about 1%; from about 1.5%; from about 2%, from about 3%,or from about 4% to about 5% arginine solution of the virus solution.The amount used can vary in different embodiments of the invention. Inyet other embodiments of the invention, the virus solution/vaccinesolutions optionally comprise potassium phosphate. In some embodiments,the solutions comprise about 11 mM potassium phosphate. In otherembodiments, the solutions comprise from about 10 mM to about 12 mMpotassium phosphate. The formulated composition can optionally containsubstantial amounts of egg allantoic fluid components (e.g. proteins andmetabolites) and/or a buffer diluent. Additionally, acceptablecompositions of vaccine can contain a buffer salt, such as a mixture ofmonobasic and dibasic sodium or potassium salts of phosphate atconcentrations ranging from, e.g., 5 to 200 millimolar or histidineand/or its salts at concentrations ranging from, e.g., 25 to 100millimolar. In preferred embodiments, sucrose is present at aconcentration ranging from about 100 millimolar to 350 millimolar.

In many virus solutions/vaccine solutions a base solution of SPG(sucrose, potassium phosphate and monosodium glutamate) is optionallyutilized. However, in some embodiments of the current invention, MSG isnot part of the virus/vaccine solution. In yet other embodiments, levelsof MSG are reduced. The amount of sucrose that is usable in theembodiments herein is variable over a wide range. Approximately 0.2 Msucrose is utilized (7% W/V) in typical embodiments, however,compositions comprising up to ca. 20% sucrose can present no detrimentaleffect on virus activity/potency. Surfactants in various embodiments ofthe compositions can comprise, e.g., Poloxamer 188(polyoxyethylene-Polyoxypropylene block copoloymer, e.g. Pluronic F68)and Tween 20 (polyoxyethylene sorbitan monolaurate) at concentrations inthe range of ca. 0.01 to 0.1% (W/V%). In some embodiments, thecombination of Poloxamer, gelatin hydrolysate and arginine is superiorto any solution containing only one of the components, each solution inturn being more stable than a solution containing none of the addedcomponents.

In yet other embodiments, steps in Group 4 (e.g., Step 15 of Table 1)comprise replacing all or part of the normal allantoic fluid (NAF) inwhich the viruses are suspended with a buffer of sucrose, potassiumphosphate and monosodium glutamate (SPG) or other simple solutions,e.g., those with reduced MSG, etc. The use of SPG in place of some orall of the NAF diluent results in greater stability of the viruses insolution. Such stability is also a novel and unexpected benefit of theembodiments of the current invention. Representative formulationsembodying some or all of the formulation attributes described above wereprepared and the stability of the component cold-adapted viruses wasevaluated. Compositions of representative formulations are shown inTable 28. The stability of the formulations at 5° C. is shown in Table29.

Various formulations of the invention were tested for their stabilityover a variety of months and temperatures. For example, Table 30illustrates 12 different formulations. Formulations 10 and 11 were basedupon formulations used for dried virus preparations. The formulations insuch tables covered a range of various components, e.g., sucrose andgelatin. Tables 31–34 show the stability of such preparations comprising4 different virus strains over six months (two sample points for each).FIG. 38 graphs the results of 4 exemplary formulations with the B/HongKong strain used. Table 35 shows compositions of additionalformulations. The compositions in Table 35 examine addition of variouscompounds to the basic composition (i.e., typically 10/2/2 meaning about10% sucrose, about 2% arginine, and about 2% gelatin) to helppotentially inhibit adverse components present in the NAF such aslysozyme, etc. The stability results of the formulations in Table 35 areseen in Tables 36 through 39 and in FIGS. 39 and 40. Tables 40 and 41and FIG. 41 a–c look at varying concentrations of citrate in theformulations (here a base formulation of about 10% sucrose, about 1%gelatin, and about 2% arginine). Formulations with citrate showed aprecipitate at about 7–8 months of storage. Tables 42 and 43 and FIG. 42a–c show a similar analysis, but with varying concentrations of EDTA.Exemplary formulations from the above examples were subjected to furthertesting which is shown in Table 44 and 45 a–d. Additional formulationswith varying concentrations of sucrose, gelatin, arginine, and EDTA,etc. are shown in Tables 46 through 48.

To further illustrate the stability of several monovalent formulationsherein, compositions comprising 60% allantoic fluid were tested forstability. Samples were stored at 5° C. and examined with FFA analysis.Biweekly sampling was done for the first two months, then monthlysampling was done to 9 months. A concentration of 60% AF will allow ahigh probability of producing VH at the necessary potency even in yearswith low titer strains. Some formulations utilizing unpurified VHexhibited sufficient stability for all strains test to almostconsistently meet a criterion of 0.5 log loss in 7 months at 5° C.Influenza strain B/Hong Kong/330/01 appeared to be the most problematicof the strains tested for stability. See Table 30 which gives percentcomposition of sucrose, arginine, gelatin and other components for the13 different formulations. FIG. 43 illustrates the stability of fourvirus strains in such formulations after 9 months. Exemplaryformulations of unpurified virus composition formulations can comprise,e.g., VH, 10% sucrose, 2% arginine, 2% gelatin; VH, 10% sucrose, 2%arginine; VH, 10% sucrose, 2% arginine, 1% dextran; VH, 10% sucrose, 2%arginine, 0.5% PVP; VH, 10% sucrose, 2% arginine, 2% gelatin, 2.5 mMEDTA; VH, 10% sucrose, 2% arginine, 2% gelatin, citrate buffer; and, VH,10% sucrose, 2% arginine, 2% gelatin, histidine buffer.

Other methods of virus/vaccine solution purification (e.g., forstabilization, etc.) involve such techniques as removal of all NAFthrough fractionization (along with addition of stabilizers) to givestability of the solutions. Various embodiments of the currentinvention, however, involve, e.g., dilution out of the NAF in which thevirus/vaccine exists. For example, in various embodiments herein, theconcentration of NAF optionally comprises from about 10% to about 60% ofthe solution. In other embodiments, NAF can optionally comprise fromabout 20% to about 50%, or from about 30% to about 40% of the solution.Such dilution of NAF concentrations allows for greater stability of thevirus/vaccine solutions, especially at desired temperatures (e.g., 4°C., from about 2° C. to about 8° C., etc.) in liquid form. Additionally,some embodiments of the invention comprise reduced NAF concentrations inconjunction with use of arginine (see above). Various formulations ofthe current invention were compared in stability with virus compositionsthat were NAF free purified formulations or that were NAF reduced (butstill NAF purified) formulations. Table 49 illustrates the formulationof a number of compositions of the invention as well a number offormulations wherein the VH was purified from the NAF various ways. Itwill be appreciated that the base formulations shown in Table 49 alsotypically comprise about 2% arginine, about 2% gelatin, about 1% PVP,about 1% dextran, about 2.7 mM EDTA, and about 100 mM histidine. Thenumbers in Table 49 correspond to the formulations displayed in FIGS.44–46.

The diluted NAF embodiments of the current invention are in comparisonto alternative stabilization methodologies, e.g., which end up with10–25% fractionated NAF or even 5% fractionated NAF or less in theirfinal formulations. However, those of skill in the art will appreciatethat the NAF present in some current embodiments does not comprise suchfractionated NAF, but is instead comprised of un-fractionated NAF. Theformulations of the invention were compared against other current virussolutions that were made from purified NAF (e.g., fractionated NAF,etc.) in terms of stability. The goal in the comparison was to reachless than or equal to 1.0 log potency loss in 12 months or less than orequal to 0.080 log/month loss in potency when stored at between 2° C.and 8° C., e.g., 4° C. The other current virus formulations comparedagainst the formulations of the invention were purified through, e.g.,fractionation, diafiltration, etc. The different formulations weretested with 3 different influenza strains: a H1N1 strain (A/NewCaledonia/20/99 or A/NC), a H3N2 strain (A/Panama/2007/99 or A/Pan orA/PA), and a B strain (B/HongKong/330/01 or B/HK) and were filled intoAccusprayers (i.e., a delivery device for FluMist®). In order to mimic alikely manufacturing process, the samples were frozen at −25° C. for atleast 6 days as an initial step.

In a first comparison, a NAF purified cold-adapted trivalent formulationwas compared in stability with an unpurified NAF formulation of theinvention. The formulations comprised 7% sucrose, 1% gelatin, 1%arginine (which are the standards for the comparing trivalent formula)and 60% AF (allantoic fluid) for the formulation of the invention. Theformulation of the invention after six months showed −0.035±0.016 forA/NC, −0.079±0.035 for A/Pan, and −0.151±0.018 for B/HK. Themeasurements for the purified composition was −0.020±0.027 for A/NC,−0.011±0.020 for A/Pan, and −0.138±0.022 for B/HK. The units above arein log FFU/month. See Table 50. Table 51 shows a comparison between apurified formulation and a formulation of the invention when theinvention formulation uses a 10/2/2 composition, see above. The highinitial potency loss observed is though to be attributed to freeze-thawand/or blending loss. Table 52 shows a similar comparison, but withhistidine in the FluMist® formulation, which gave rise to a betterstability with no initial potency loss observed.

FIG. 44 illustrates the initial potency loss (freezing and/or blendingloss) seen above is exclusively associated with phosphate bufferedformulations. No initial potency loss was observed with histidinebuffer, which exerted a positive impact on stability. The formulationsshown in FIG. 44 are those listed in Table 49. FIG. 45 illustrates a“global” picture of the stability slopes of the formulations of Table 49after 6 months. As can be seen the histidine buffered 10/2/2 formulationexhibited the best combination of stability and meeting the target goal.See above. FIG. 46 gives a different view of similar data (i.e., weekrather than month). FIG. 47 illustrates a second study which producedresults illustrating the stability of a 10/2/2+histidine formulationwith either gelatin (L106) or PVP/EDTA (L104). As can be seen from thefigure, the replacement of gelatin with PVP/EDTA produced stabilityalmost as efficiently as the inclusion of gelatin. FIG. 48 examines theoptimal pH of a histidine-based 10/2/2 formulation of the invention. Ascan be seen, pH 7.0 comprises a preferred embodiment. Ranges of pH fromabout 6.8 to about 7.2 for these 100 mM histidine 10/2/2 formulationsare also included embodiments of the invention. FIG. 49 showsexamination of preferred embodiments of sucrose concentration inembodiments of the invention. Some preferred embodiments comprise about10% sucrose, while others comprise about 7%. The basic formulation inFIG. 49 comprises the 10/2/2 above, with the addition of sucrosehistidine. In the various embodiments illustrated herein, someembodiments comprise histidine as a buffering additive and/or arginineas a stabilizer and/or dextran and/or PVP in place of gelatin.

Other embodiments of the current invention are optionally stabilizedthrough use of ultrafiltration/concentration of the virus/vaccinesolution. Such ultrafiltration is typically an alternate means ofachieving solution stability as opposed to reductions/dilutions of NAF.For example, in some situations if the titer or potency of a particularstrain/solution is low, then ultrafiltration can optionally be used inplace of NAF dilution (which could act to further reduce thetiter/potency of the solution). The ultrafiltration in Groups 4 steps isslightly different from the microfiltration as described above. In theearlier Group the filtration was for, e.g., sterility whereas in thecurrent Group the filtration concerns stability, etc. and the virusesare kept during the filtration. See above.

Potency Assays

In some embodiments herein, the potency measurement for thevirus/vaccine is performed by a cell-based ELISA (i.e., Cell-basedELISA, or CELISA, for Potency Measurement of FluMist-a live, attenuatedinfluenza virus vaccine, or for other such vaccines). Such method is asimpler and faster alternative to the more traditional Median TissueCulture Infective Dose (TCID₅₀) assay, for potency measurement of livevirus. Briefly, confluent monolayers of Madin-Darby Canine Kidney (MDCK)cells in 96-well microtiter plates are infected with sample containinglive virus, fixed with formalin 16–18 hours post-infection and reactedwith influenza virus-specific monoclonal antibody (Mab). Virus antigenbound Mab is then detected using anti-mouse IgG˜Peroxidase andperoxidase substrate to develop soluble colored product, the opticaldensity (OD) of which is measured spectrophotometrically. Those of skillin the art will be familiar with epitopes/antigens shared by varioussubtypes of influenza strains (e.g., various HA, etc.). The potency oflive virus in a sample is calculated from a standard curve generatedusing live influenza virus calibrators with known log₁₀ TCID₅₀ valuesobtained with a validated TCID₅₀ potency assay. CELISA is shown to belinear (r² greater than or equal to 9.95) in the range 4.9–6.7 log₁₀TCID₅₀. Between-day, between-analyst, between-plate, within plate(residual) variability (Standard Deviation in log₁₀TCID₅₀) were 0.06,0.02, 0.05 and 0.03 respectively. The potency of several vaccine andwild-type influenza A/H1N1, A/H3N2 and B strains measured by CELISA arecomparable (±0.3 log₁₀TCID₅₀) to the potency measured in parallel by thevalidated TCID₅₀ potency assay. CELISA is capable of measuring potencyof up to 10 samples/plate in 2 days in contrast to 2 samples/plate in 6days for the validated TCID₅₀ potency assay. CELISA is optionally usedin place of, or in addition to other methods of potency assay (e.g., FFAand TCID₅₀, see, below).

The Median Tissue Culture Infective (or Infectious) Dose 50% (TCID₅₀)assay (see, below for more details) is a widely used method for thepotency measurement of live virus and live virus vaccines. However, insome embodiments herein, Cell-based ELISA (CELISA) is optionally used asa simpler and faster alternative to the traditional, long and laborintensive TCID assay to measure potency of influenza virus in FluMist, alive, attenuated vaccine (or in other similar vaccines).

In other typical embodiments, potency assays of the virus solutionsoptionally comprise fluorescent focus assays (FFA) as opposed to commonTCID₅₀ assays which are used in the art. Such FFAs have the addedbenefit that they are more amenable to automation, thus, allowing higherthroughput of vaccine production. TCID₅₀ assays usually measure thequantity of a virus suspension or solution that will infect 50% of aparticular cell culture. The measurement gives accurate results, but isslower than FFA and thus can use up valuable time in the production ofvaccines. FFA assays typically use type and/or subtype (or evenuniversal antigen) specific anti-influenza antibodies (typically anti HAantibodies) to detect virus antigens in infected cells. In uses whereinthe antibodies do not cross react with different types/subtypes ofinfluenza they can be used to quantitate the separate virus types inmulti-virus preparations (e.g., trivalent vaccine formulations). FFAassays can also be used as identity tests for specific strains. Those ofskill in the art will be quite familiar with FFAs and their use invirus/vaccine testing.

Fluorescent focus assays, on the other hand, do not rely on theinduction of cell death (either in the infected cells or the indicatorcells). Instead, they use antibody staining methods to detect virusantigens within infected cells in a cell culture monolayer. Theseinfected cells are then visualized and quantified using a fluorescentlabel on the virus-specific antibody. Typical FFAs of the currentinvention use, e.g., type and subtype specific anti-influenza HAantibodies to visualize virus antigens in infected cells.

In other embodiments, the FFAs (and optionally other assays herein)optionally use a universal reagent (or universal antigen) which is notspecific for specific type/subtype influenza antigens, but is insteadspecific for a generalized influenza antigen. Therefore, the universalreagent is optionally useful for FFAs for myriad different screeningsand type/subtype specific antibodies do not have to be developed andcreated each time a different virus is assayed.

Other embodiments herein comprise viral potency determination using acell-based fluorometry assay (CFA). While FFA assays are quite useful inmany embodiments, CFA assays are preferentially used in otherembodiments. While the image processing and readout of FFA assays can becapped at about 20 plates/person/day (or about 5 plates/hour imageprocessing), the image processing and readout from CFA assays can be upto about 4 times faster. Also, while FFA titers can differ from TCID₅₀titers for influenza B strains, CFA titers have not shown significantdifferences from TCID₅₀ (or FFA) titers due to the use of assay standardor calibrators. In brief, the CFA assay measures infectious influenzaviruses in MDCK cells grown in 96 well plates. As with FFA, CFA detectsviral protein expression resulting from viral infection of MDCK cellsduring the first infection cycle. CFA assays utilize calibrator or assaystandard for titer calculation. For CFA reagents, typical antibodyreagents can comprise: primary antibodies specific to HA or A strainsand B strains (for influenza) and secondary antibodies of, e.g., goatanti-mouse IgG conjugated with Alexa 488. Assay standards for CFA caninclude virus harvest of the same strain as the samples to be testedwith a known FFA or TCID titer. Assay references for CFA can include,virus harvest with known FFA or TCID titer and known linear slope (doesnot have to be the same strain as the samples to be tested). Sampleprimary antibodies can include, e.g., those specific for A/H1N1 orA/H2N2 strains (from, e.g., Takara) at, e.g., a working dilution of1:2000; those specific for A/H3N2 strains (from, e.g., Takara) at, e.g.,a working dilution of 1:1000; and, those specific for B strains (from,e.g., Chemicon) at, e.g., a working dilution of 1:1000. A typical CFAassay procedure can comprise virus inoculation followed by 18 hoursincubation at 33° C. followed by fixation and incubation at roomtemperature for 15 minutes. A primary antibody incubation for 60 minutesat 37° C. is then followed by a secondary antibody incubation for 60minutes at 37° C. The plates are then read with a fluorometer and thedata analyzed. In some embodiments of CFA, the infection level of awell, etc. is determined via protein expression (as opposed to typicalFFA assays where the number of cells infected are measured). Those ofskill in the art will be aware of typical FFA assays and fluorometry andsimilar concerns applicable to CFA assays.

Semi-Automated TCID₅₀ Assay

As stated above, some embodiments of the current invention compriseTCID₅₀ assays and various modifications, etc. thereof. For example, someembodiments comprise a semi-automated version such as illustrated by thefollowing descriptions.

A comparison of Manual and Semi-Automated Median Tissue CultureInfective Dose (TCID₅₀) assays for Potency Measurement of a live,attenuated influenza virus vaccine, e.g., FluMist®, or other similarvaccines, is given in this section. The TCID₅₀ potency assay isoptionally used for potency measurement of FluMist or other similarvaccines. A semi-automated TCID₅₀ potency assay is described in whichtwo labor-intensive steps of the validated manual potency assay areimproved. These are (i) use of an automated pipetting station for sampledilutions and infection of MDCK monolayers in place of multiple manualrepeating dilution steps, and (ii) use, 6-days post-infection, of a96-well plate reader to measure spectrophotometrically the product ofMTT, a vital dye (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) widely used as an indicator of cell health/viability, in placeof manual observation under a light microscope of each of the 96 wellsof all assay plates in order to assess the presence of influenza virusinduced cytopathic effect (CPE) in MDCK cells.

The semi-automated TCID₅₀ potency assay used in some embodiments hereinwas developed and validated to demonstrate precision (repeatability:<0.25 log₁₀TCID₅₀; intermediate precision: SD_((Day))<0.3 log₁₀TCID₅₀;SD_((Analyst)) and SD_((Instrument))<0.4 log₁₀TCID₅₀; andreproducibility at the 90% Confidence Interval ±0.3 log₁₀TCID₅₀),linearity, accuracy and range (Slope 1±0.1). The semi-automated TCID₅₀potency assay using an automated pipetting station and MTT dye was shownto provide equivalent results to the validated manual TCID₅₀ potencyassay (at the 90% Confidence Interval ±0.3 log₁₀TCID₅₀). In brief, theresults herein provide support for the use of a pipetting station andMTT dye to measure the potency of influenza virus in, e.g., FluMist®productions. These improvements also increase the testing throughput.

Infectivity/Potency (Monovalent) Assay Validation

A semi-automated version of the current manual method for potencydetermination of monovalent influenza strains is optionally used formanufacture of FluMist™ vaccine and other similar vaccines. Thesemi-automated potency assay incorporates automation of the platewashing and serial dilution steps, and an automated dye-based detectionof the virus induced cytopathic effect (CPE) to replace the manualmicroscopic detection. Automation of the plate washing and serialdilution steps allows greater assay throughput, and reduces the risk ofrepetitive motion injuries for Quality Control analysts performing thisassay. Automated dye-based detection of the virus induced CPE enhancesassay consistency and throughput by elimination of the microscopicdetection.

Some steps/aspects of the semi-automated assay are similar to moretraditional TCID₅₀ assays, while other steps/aspects are quitedifferent. The assay steps include preparation, incubation and washingof assay plates containing Madin-Darby canine kidney (MDCK) monolayers,infection and post-infection incubation of the assay plates, andcalculation of the potency based on the number of CPE positive wells andsample testing configuration in both assays. The semi-automated potencyassay has been validated and the performance of the assay demonstratedto be comparable to the current manual assay by the inventors andcoworkers. This semi-automated assay optionally serves as the primarymethod for the infectivity/potency measurement of expanded wild-typeinfluenza (eWT), Master Virus Seed (MVS), Manufacturer's Working VirusSeed (MWVS), and Virus Harvest (VH) samples. The manual assay isoptionally used as a backup in a situation where the semi-automatedassay cannot be performed; i.e. in the case of prolonged equipmentdowntime.

The traditional median tissue culture infectious dose (TCID₅₀) assay isa cell-based method that measures infectious cytocidal virions. MDCKcells are cultured in 96-well plates, and confluent monolayers areinoculated with serial dilutions of a virus sample. Replication of virusin the MDCK cells results in cell death. The progeny virus infects othercells, resulting in the eventual destruction of the monolayer. The CPEresulting from infection is allowed to develop during an incubationperiod of six days. The individual wells are read microscopically, todetermine the presence of CPE in each well. Four individualdeterminations are performed on each of three days by this procedure,and the results of all 12 titrations are averaged in order to produceone test result. In addition to samples, each analyst analyzes onemonovalent control, also with four replicate determinations on each, ofthree days.

The manual assay is labor intensive and has limited sample throughput.Each individual determination involves numerous cell washing and serialdilution steps, which are performed using a manual pipettor. Each wellin the 96-well assay plate has to be microscopically scored for thepresence or absence of CPE. Multiple plate washing and dilution stepspose a repetitive motion injury risk for analysts. In addition, themicroscopic reading of each individual well in the 96-well plates isfatiguing, which limits the number of analyses that can be performed byeach analyst to about 20 plates per day. One test result is obtained asan average of twelve determinations over a three-day test period, andeach analyst performs one monovalent assay control in addition tosamples. This limits the assay throughput to 9 samples per analyst per3-day testing period (average of 3 samples per analyst per day). Becauseeach individual sub-lot (about forty to fifty sub-lots per lot) ofmonovalent influenza vaccine harvest is tested by this assay, thelimited throughput may limit capacity for full-scale commercializationof vaccines (e.g., FluMist™ vaccine, etc.).

Automation of the traditional assay, specifically the plate-washing andpipetting steps, and MTT(3-[4,5-Dimethylthiazol-2-yl]2,5-diphenyl-tetrazolium bromide) dye-baseddetection of CPE, results in development of a Semi-Automated TCID₅₀Potency Assay for Influenza Virus Monovalent. The semi-automated assayuses the Skatron™ Cell Washer for the washing steps, where the debrisand spent media are removed from the cell culture plates and replacedwith fresh media. The Matrix SerialMate® multichannel pipetting stationis used to perform the sequential 10-fold dilutions of the virus, andfor transfer of the diluted samples onto the cell culture monolayers in96-well assay plates. Of course, other devices which perform similarfunctions are optionally substituted herein and specific mention ofparticular brands or types of devices should not be construed aslimiting unless specifically indicated to be so. After the six-dayincubation period, the 96-well assay plates are then incubated for sixhours with MTT dye, which is a widely accepted indicator of cellmetabolism and viability. During the incubation period, intact andhealthy cell monolayers process the dye to form the insoluble purpleformazan product, which accumulates intracellularly. In wells where thecell monolayer is destroyed, no dye product is formed. A solubilizingsolution of 0.01 N Hydrochloric Acid, containing 20% of the surfactantsodium dodecyl sulfate (SDS) is then added, and the plates incubatedovernight to dissolve the insoluble dye product. The absorbance at 570nm is measured to quantify the purple formazan dye product. Theabsorbance reading is processed using a Microsoft Excel™ Macro program(or other similar program), to identify and count the CPE positive ornegative wells and calculate the TCID₅₀ titer. Wells containing intactcell monolayers show a higher absorbance when compared to apre-determined cut-off value, and are identified as CPE negative,whereas CPE positive wells show absorbance readings below the cut-offvalue (see FIG. 50). The number of wells showing CPE at each dilution isthen used to calculate the titer (log10 TCID₅₀/mL) based on the Karbermodification of the Reed-Muench method. The automation of the cellwashing, serial dilution and virus inoculation steps, and the MTTdye-based CPE detection are described in detail below.

Automation of Cell Washing Steps Using Skatron™ Cell Washer

In a manual assay, plates containing MDCK cell monolayers in 96-wellplates are washed twice prior to inoculation with the diluted virussamples. Spent medium containing waste products and fetal bovine serum(FBS) from the four-day cell incubation is removed and replaced withfresh virus growth medium (VGM) without FBS. The cells are thenincubated at 33±1° C. and 5±1% CO₂ for at least 10 minutes, then the VGMis removed and replaced with fresh VGM a second time. For each washingstep, individual plates are inverted onto clean paper towels and gentlyblotted to remove media from the wells, and then each well is refilledwith 200 μL of fresh VGM using a hand-held multichannel pipettor. Thisprocess is labor-intensive and time consuming when large numbers ofplates are processed.

The Skatron™ Skanwasher (Series 300, Model 12010) is amicroprocessor-controlled 96-channel cell washer, which performs thesewashing steps automatically. The Skanwasher is small enough to fit in a6-foot laminar flow biosafety hood. Automation of cell plate washingsteps using the Skatron™ Skanwasher, involves a wash program where thespent media are aspirated from the plates, then fresh VGM is dispensedinto the empty wells. Individual plates are loaded into the Skanwasher,then removed to a 33±1° C. and 5±1% CO₂ incubator at the end of the washcycle. The plates are incubated for a minimum of 10 minutes, then loadedonto the Skanwasher for the second wash, after which they aretransferred back into the incubator. The performance of the Skatron™Skanwasher in these wash steps is shown to be acceptable for use in thecell washing steps. The dispensing precision for the 200 μL volume isassociated with a CV<10%, and the dispensing accuracy is within 10%. Theresidual volumes for the aspiration step are less than 1%. Thus theSkatron™ Skanwasher provides acceptable performance, while improving theease of use and throughput efficiency of the cell-washing step. Again,it will be appreciated that similar devices capable of performancewithin the same standards are also optionally used herein.

Automated Serial Dilution and Virus Inoculation with Matrix SerialMate®Multichannel Pipetting Station

The serial dilution and the virus inoculation steps of the traditionalmanual TCID₅₀ assay are carried out by hand-held multi-channelmicropipettes. The serial dilutions are carried out in two steps. Thefirst set of five serial dilutions is carried out in a 0.5 mL dilutionblock, and then the appropriate dilution from the first block istransferred to a 2 mL dilution block, for the final five serialdilutions. It is crucial that these serial dilutions be carefullyexecuted, because pipetting errors at any one dilution may be propagatedand magnified through the subsequent series. The subsequent virusinoculation step involves repetitive pipetting of diluted virus intomultiple rows or columns of an assay plate containing confluent cellmonolayers. Prolonged use of the hand-held multichannel micropipettesused to provide the necessary accuracy for these tasks can lead tosevere muscle fatigue and tendonitis, which limits the number of plateseach analyst can perform in one day and, thus, the throughput of theentire process.

Use of the Matrix SerialMate® pipetting station for the serial dilutionand virus inoculation steps improves the ease of use and throughput ofthe assay, and reduces the occurrence of operator injuries, whileproviding the necessary precision and accuracy for these tasks. TheMatrix SerialMate® pipetting station is a bench top liquid handlingstation equipped with a 12-channel nozzle head which can aspirate anddispense volumes in the range of 5 μL–225 μL. The unit is small enoughto fit in a standard 4- or 6-foot biosafety cabinet and is easy to use.The Matrix SerialMate® provides precision better than 0.5 μL, andaccuracy better than 1.0 μL for delivery volumes of 5 μL–225 μL. Thiscorresponds to a precision better than ±1.7% and accuracy better than±3.3% for the 30 μL delivery volume used in the serial dilution steps.The comparability of assay results obtained using the automated assayand the current manual assay is confirmed as described below. Again, itwill be appreciated that similar devices capable of performance withinthe same standards are also optionally used herein.

Description of MTT Dye-Based Detection

The final step in a TCID₅₀ assay is the detection of CPE andquantitation of the virus. With the current (manual) TCID₅₀ assay, theindividual wells are read microscopically, to look for signs of CPE ineach well. These signs include areas of foci, partial or completecollapse of the cell monolayer, and the presence of rounded and darkenedcells on top of the destroyed cell monolayer. It has been observed thatsignificant eye strain sets in as the analyst counts large numbers ofplates, setting the practical limit for the number of plates which maybe counted by one operator to about 20 plates. This step is ratelimiting to the throughput of the manual assay.

Tetrazolium dyes are widely used as cell viability indicators. The mostcommonly used dye is yellow MT dye. Viable cells, which possess activemitochondria, will reduce MT dye to an insoluble purple formazanproduct, which can be detected at 570 nm after a solubilization step. InCPE positive wells where the large majority of cells have beendestroyed, little or no dye product is formed, and a much lowerabsorbance is observed.

In a semi-automated TCID₅₀ assay, after the infection and six-dayincubation of the plates, the spent medium is removed, 100 μL of asolution of 0.5 mg/mL MTT dye in fresh virus growth medium is added toeach well of the 96-well plates, and the cells are incubated at 37±1° C.and 5±1% CO₂ for six hours. The dye product is solubilized by overnightincubation at 37±1° C., following addition of 100 μL of a solubilizingreagent (20% SDS in 0.01 N HCl), then the absorbance at 570 nm due tothe purple formazan dye product is measured with a plate reader. Theabsorbance data is transferred to a validated Microsoft Excel™ Macro (orother similar program) that converts the absorbance readings to a CPEcount based a pre-established cut-off value. Wells containing intactcell monolayers yield a higher absorbance when compared to apre-determined cut-off value, and are identified as CPE negative. CPEpositive wells show absorbance readings below the cut-off value. Thenumber of wells showing CPE at each dilution is then used to calculatethe titer (log10TCID₅₀/mL) based on the Karber modification of theReed-Muench method.

The automated dye-based detection enhances the consistency of the CPEreadout and increases the assay throughput. The comparability of thedye-based detection to the manual microscopic CPE detection is ensuredby extensive studies where the assay was run with different vaccine andwild type virus strains, and with plates prepared with different cellpassage numbers and seeding densities. In these studies the plates wereread first by manual microscopic examination, and then by dye-basedabsorbance detection. The results from these studies were analyzed todetermine a universal absorbance cut-off, which provided comparable CPEcounts by both detection methods. This universal cut-off value of 0.5254for the absorbance at 570 nm was confirmed by a detailed study (see,below), in which 9 different analysts performed assays on threedifferent instruments, over 6 assay days, using a total of 573 assayplates. The presence or absence of CPE in each well (80 virus inoculatedwells per plate, for a total of 45,840 wells) was read first by manualmicroscopic examination, then by dye-based absorbance detection.

FIG. 50 shows a histogram derived from plotting the absorbance readingsfrom the wells, versus the frequency of the values (number of wells readat that absorbance value). The frequency determination shows that theabsorbance cut-off value of A570=0.5254 is located in the left-most tail(probability=0.007%) of the distribution of the CPE negative wells, andthe right-most tail of the distribution of CPE positive wells(probability=0.02%). Comparison of the CPE detection of each well byboth methods using this cut-off value, showed a one-to-onecorrespondence in most wells (45279 of 45840, 98.78%) for identificationas either CPE positive or CPE negative, both by dye-based detection, andby microscopic examination.

Validation of Semi-Automated Potency Assay

The Semi-Automated median tissue culture infectious dose (TCID₅₀)potency assay for analysis of monovalent influenza vaccine virus isintended for the infectivity/potency measurement of expanded wild-typeinfluenza (eWT), master virus seed (MVS), manufacturer's working virusseed (MWVS), and virus harvest (VH) samples. The assay was validated todemonstrate the precision (repeatability, intermediate precision andreproducibility), linearity, accuracy, and range of a Semi-AutomatedTCID₅₀ assay, and show that it provides comparable results to a manualTCID₅₀ assay. Validation tests were carried out with three differentmonovalent vaccine strains, chosen to include one Type A/H1N1, one TypeA/H3N2 strain, and one Type B strain. The assay validation was carriedout by two separate groups in different laboratories, to demonstratelaboratory-to-laboratory reproducibility. The precision (between-testvariability), linearity, accuracy and range of a semi-automated assayare compared with those observed for a manual assay in Table 53.

The between-test standard deviation (SD) of the semi-automated assay,was evaluated from six tests performed on each of three vaccine strains,by the same analyst group, on the same pipetting station (each testresult is obtained by averaging 12 determinations obtained over threedays). The acceptance criterion for the between test variability of thesemi-automated assay was 0.25 log10 TCID₅₀/ml, which is the half-widthof the 95% confidence interval for a single test result based on thehighest observed variability (0.11 log10 TCID₅₀units) of the manualassay. The actual SD values obtained with the semi-automated assay, forthe three strains, ranged between 0.06–0.09 log10TCID₅₀/mL. These valuesare within the acceptance criterion of SD<0.25 log10 TCID₅₀ units andare comparable to the between-test variability (0.07 to 0.11 log10TCID₅₀ units) observed for the manual TCID₅₀ assay, from nine repeattests performed on three independent lots of each of three strains.

The assay was demonstrated to be linear over a 105-fold dilutionrange(titer range of 4.2–9.3 log10TCID₅₀/mL), by showing that therelationship of the calculated and measured TCID₅₀ titer passed a testfor lack of fit to a linear model at the 1% significance level. Theassay was accurate, with slopes of 1.00–1.02 for the three strains,which were all within the acceptance criterion of slope of 1±0.1. Thelinearity, accuracy and range of the semi-automated assay are comparablewith the manual assay. See Table 53.

Intermediate precision of the semi-automated assay was demonstrated byfitting a random effects model to a set of 18 tests obtained by twoanalyst groups over nine different assay days, on one type A and onetype B vaccine virus strain. The measured standard deviation ranges forthe between day variability (SD(day)), between analyst group variability(SD(analyst)), and between instrument variability (SD(instrument)) wererespectively 0.04–0.08, 0.14–0.16, and 0.000–0.03, which met theacceptance criteria of SD(day)<0.3, SD(analyst)<0.4, andSD(instrument)<0.4.

The inter-laboratory reproducibility of the assay was demonstrated bycarrying out assays on one Type A/H1N1one TypeA/H3N2 and one Type Bstrain in two different laboratories. The acceptance criterion forlaboratory-to-laboratory reproducibility required the two sided 90%confidence interval for the difference in the mean results from the twolaboratories to be within ±0.3 log10TCID50/mL. This acceptance criterionwas met, with the lower and upper bounds of the 90% confidence intervalsof greater than −0.05 and less than +0.15, respectively, for all threestrains.

A detailed statistical comparison was performed to demonstrate thecomparability of the Manual and Semi-Automated assays. Two vaccinestrains, one Type A/H1N1 (A/New Caledonia/20/99) and one Type B(B/Yamanashi/166/98), were assayed manually to obtain 18 test results oneach strain. The data for all 18 test results obtained manually for eachstrain, were pooled and compared with the pooled test results from theprecision and intermediate precision studies carried out forsemi-automated test (18 test results per strain). The Proc Mixed methodin SAS was used to estimate the between method mean difference and its90% confidence interval (CI). The acceptance criterion was that the 90%CI must be within ±0.3 log10TCID₅₀/mL, i.e. the lower bound (LB) of the90% CI must be greater than −0.3, and the upper bound (UB) must be lessthan 0.3. The results are presented in the assay validation report andsummarized in Table 54, below. As may be seen from the results in, thetwo-sided 90% confidence intervals were within the acceptance criteriaof ±0.3 log10TCID₅₀/mL for both strains, with actual estimates of thelower and upper bounds ranging between−0.05 and 0.10 log10TCID₅₀/mL.

Thus, in summary, while Manual TCID₅₀ Potency Assay for Influenza VirusMonovalent, is the traditional validated assay for theinfectivity/potency measurement of monovalent influenza vaccine strainsin expanded wild-type influenza (eWT) Master Virus Seed (MVS),Manufacturer's Working Virus Seed (MWVS), and Virus Harvest (VH)samples, it is a labor-intensive method involving numerous manualpipetting steps, which pose a repetitive motion injury hazard toanalysts. In addition it uses a manual microscopic CPE readout, whichlimits the assay throughput to 3 tests per test day per analyst.Automation of the plate-washing and manual pipetting steps, andsubstitution of MTT dye-based detection of CPE for the manualmicroscopic readout can result in development of a Semi-Automated TCID₅₀Potency Assay for Influenza Virus Monovalent. The implementation of thesemi-automated assay, for testing of monovalent materials optionallyincreases the assay throughput 2–3 fold, and allows practicalcommercialization of vaccines such as FluMist™ Vaccine at theanticipated level of doses for market. An additional benefit is alowered risk of repetitive motion injuries for Quality Control analysts.

The semi-automated assay has been validated to demonstraterepeatability, intermediate precision, linearity, and accuracy for assayof viral materials in the titer range of 4.2–9.3 log10TCID₅₀/mL in onegroup. The assay was also validated to demonstrate inter-laboratoryreproducibility with another group.

A detailed statistical comparison of results obtained by using both thesemi-automated assay and the manual assay, for repeated potencymeasurements of one Type A and one Type B influenza strains, also showedthat the two assays yield comparable results. Thus, the semi-automatedassay is demonstrated to be comparable to the manual assay for use inthe potency measurement of expanded wild-type influenza, FluMist™ mastervirus seed (MVS), manufacturer's working virus seed (MWVS), and virusharvest (VH) samples.

Universal Cutoff Value of CPE in Semi-automated TCID₅₀ Assays

In yet other embodiments herein other variations and modifications ofTCID₅₀ assays are employed to determine potency of vaccine/viruses. Onesuch modification is the confirmation of the universal cutoff value forthe assessment of CPE for the TCID₅₀ SemiAutomated Potency Assay forinfluenza virus monovalent. The “SemiAutomated TCID₅₀ Potency Assay forInfluenza Virus Monovalent” (see above) uses the viable cell dye MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to scorethe cytopathic effect (CPE) in infected monolayers of MDCK cells. Toreliably determine virus potency values using the MTT colorimetricendpoint to detect the number of CPE-positive wells, it is useful toestablish an absorbance cutoff value that reproducibly distinguishesbetween CPE-positive and CPE-negative wells. As described in other workby the inventors, a “universal cutoff” absorbance (A570) value of 0.5254has been determined. In the “SemiAutomated TCID₅₀ Assay for InfluenzaVirus Monovalent,” a well is considered CPE-positive with an absorbancevalue of A570<0.5254; CPE-negative wells have absorbance values ofA570>0.5254.

The data summarized in this section validates the universal cutoff valuedetermined previously. The extensive testing of cold-adapted influenzastrains, A/New Caledonia/20/99 (type A/H1N1), A/Sydney/05/97 (typeA/H3N2), and B/Yamanashi/166/98 (type B) not only generate reinforcingdata for the universal cutoff assignment, but allow comparisons amonganalysts and instruments. The data presented herein substantiate therobustness, reproducibility and reliability of the Semi-Automated TCID₅₀assay and demonstrate comparability to the validated manual potencyassay. Thus, illustrating the strength of embodiments comprising thesemeasurements.

As explained above, the median tissue culture infectious dose (TCID₅₀)assay is a cell-based assay that measures the potency of infectiouscytocidal virions. Serial dilutions of a virus sample are added toconfluent monolayers of Madin-Darby canine kidney (MDCK) cells grown in96-well plates. Replication of the virus in the MDCK cells affects cellmetabolism, eventually resulting in the release of progeny virus intothe culture supernatant and cell death. The progeny viruses in turninfect other cells, resulting in the eventual destruction of themonolayer. The cytopathic effect (CPE) resulting from the infection isallowed to develop during an incubation period of six days. After thisperiod of time, MTT is used to detect the presence or absence of CPE inthe cell monolayer. Vital dyes like MTT have been used extensively asindicators of cell health and viability in cell-based bioassays (see,e.g., Denizot et al., J. Immun. Methods (1986) 89:271–277; Gerlier etal., (1986) J. Immuno. Methods 94:57–63, Heeg, et al., J. Immuno Methods(1985) 77:237–246, Mooseman J. Immuno. Methods (1983) 65:55–63, Tada, etal., J. Immuno. Methods (1986) 93:147–165, and Vistica, Cancer Research(1991) 51: 2515–2520 ). Wells containing an intact monolayer of viablecells (CPE-negative) process the dye to a purple formazan dye productand yield a high absorbance value at 570 nm (A₅₇₀). In contrast,CPE-positive wells are marked by lower absorbance values due to thepartial or complete monolayer destruction caused by the virus. Toreliably determine virus potency values using a colorimetric endpoint todetect the number of CPE-positive wells, it is useful to establish anabsorbance cutoff value that reproducibly distinguished betweenCPE-positive and CPE-negative wells. Used in conjunction with theuniversal cutoff value, absorbance values from virus test samples arescored CPE-positive or CPE-negative. The number of CPE-positive wells isused to calculate the virus titer (log10 TCID₅₀/mL).

Work by the inventors and coworkers provides an initial recommendationof the universal cutoff value based on two studies performed overseveral days by multiple analysts with three influenza virus strains. Asdescribed, a well was considered CPE-positive with an absorbance valueof A₅₇₀<0.5254; CPE-negative wells had absorbance values of A₅₇₀≧0.5254.The current section describes additional experiments designed tovalidate the absorbance cutoff value previously determined. Multipleanalysts from two independent assay groups determined the potency ofthree reference virus strains using the SemiAutomated TCID₅₀ assay. Asdescribed, CPE assessed by the validated manual method of microscopicexamination was considered the “gold standard” and compared to the CPEdetermined by MTT. The extensive testing of A/New Caledonia/20/99 (typeA/H1N1), A/Sydney/05/97 (type A/H3N2), and B/Yamanashi/166/98 (type B)not only generated reinforcing data for the universal cutoff assignment,but allowed comparisons among analysts and instruments. The datapresented herein substantiate the robustness, reproducibility andreliability of the SemiAutomated TCID₅₀ assay demonstratingcomparability to the validated manual potency assay.

The development of the SemiAutomated potency assay required the use ofreference virus strains with known potency values previously determinedusing a validated manual potency assay. The reference cold-adapted virusstrains were as follows: A/New Caledonia/20/99, a type A/H1N1 virus;A/Sydney/05/97, a type A/H3N2 virus; and B/Yamanashi/166/98, a type Bvirus. The cold-adapted control virus strain A/Sydney/05/97, was used toconfirm system suitability.

The method for the SemiAutomated TCID₅₀ Potency Assay for InfluenzaMonovalent has been developed by the inventors and coworkers as well asthe overall assay configuration for half-plate replicates, as well asthe visual CPE scoring method. See above. Briefly, confluent monolayersof MDCK cells in 96-well plates are washed twice with virus growthmedium (VGM) using a Skatron™ Cell Washer. Serial ten-fold dilutions ofvirus samples are prepared in VGM containing TPCK-trypsin using aMatrix™ SerialMate Pipetting Station and 96-well dilution blocks. Thelast five serial dilutions (10-5 to 10-9) are transferred to MDCK cellplates to achieve final virus concentrations ranging from 10-6 to 10-10relative to the initial starting titer. This format derives two potencydata points from each plate. Since each sample is assayed on two plates,four replicate potency values are obtained. The 16 control wells (platecolumns 6 and 7) receive virus-free VGM and serve as cell controls.After a 6-day incubation (33±1° C. with 5±1% CO₂) all wells are examinedusing a microscope and were scored for the presence or absence of CPE.Thus, a well is scored CPE-positive if the monolayer contained anyevidence of virus destruction. Conversely, the monolayer in aCPE-negative well was completely intact.

After visually scoring the monolayers on the plates for CPE, the mediais discarded and MTT (0.5 mg/mL), (US Biochemical Corporation,Cleveland, Ohio), prepared in phosphate buffered saline is dispensed toeach well (100 μL/well). The monolayers are incubated with MTT for 6±0.5hours at 37±1° C. with 5±1% CO₂. Solubilization buffer (100 μL of 20%SDS in 0.01N HCl) is added to each well and the plates are incubated for16 to 20 hours at 37±1° C. in an environment of 5±1% CO₂. The absorbancevalues at 570 nm are determined using a PerkinElmer-Wallac 1420Multilabel Counter Spectrophotometer and were exported into a Microsoft™Excel macro; a program used to calculate virus titer (log₁₀ TCID₅₀/mL)from the number of CPE-positive wells.

Acceptance criteria are applied to the embodiment within this section.Accordingly, a plate was considered valid if not more than one of thesixteen cell control wells on each plate showed visual evidence of CPE,cell toxicity, or microbial contamination. In addition, for eachhalf-plate to be valid, no less than 5 and no more than 36 wells had tobe scored CPE-positive. Finally, both the mean and standard deviation(SD) of the four replicate TCID₅₀ titer values obtained for themonovalent virus control sample (A/Sydney/05/97) had to be within thequalified range reported in the qualified control certificate.

Estimates of sensitivity and specificity were calculated based on therelationship between the “gold standard” CPE and MT-assessed CPE shownbelow. TP denotes “true positive”, FP is “false positive,” FN is “falsenegative,” and TN is “true negative.” Therefore, “all positives” wouldbe the sum of TP+FN, and “All negatives” would be the sum of FP+TN. SeeTable 55. The calculations are: Sensitivity for each replicate=(TP)/(AllCPE positive) and Specificity for each replicate=(TN)/(All CPE negative)

In order to perform an instrument to instrument comparison, potencyvalues were determined for three reference virus samples by six analystsin a first group using the SemiAutomated TCID₅₀ assay. Two sets of labinstruments AZ-039 and AZ-040 were used over three days. A second groupsof testers used one instrument system, AZ-036. Three analysts from thatgroup performed the SemiAutomated TCID₅₀ assay on three days using thethree reference virus samples.

In order to perform an analyst to analyst comparison, each analyst inthe testing (Analyst # 1–6) in Group 1 performed a SemiAutomated TCID₅₀potency assay on the three reference strains using instrument AZ-039over three days. In the second group, each of the three analysts(Analyst # 7–9) performed the SemiAutomated TCID₅₀ potency assay withthe same three reference virus strains over three days using instrumentAZ-036.

To reliably distinguish between CPE-positive and CPE-negative wells inthe SemiAutomated TCID₅₀ potency assay using Mu, a universal cutoffvalue was statistically determined. In an effort to validate the use ofthis cutoff value, further independent evaluation by the two groupsgenerated an additional 45,840 absorbance values. The results arepresented below.

Sensitivity and specificity measurements were calculated using themanual microscopic method as the reference standard. The combined datafrom the two groups (n=45,840) are shown in Table 56. Using therecommended cutoff value of 0.5254 resulted in a sensitivity of 98.45%and a specificity of 99.12%. In addition, the data from the second groupfor sensitivity and specificity determinations were 99.15% and 99.99%,respectively. Likewise, the data from the first groups, for sensitivityand specificity were 98.13% and 98.71%, respectively. All data above(>95% sensitivity and >95% specificity) correlates with the datadetermined wherein a sensitivity of 99.05% and a specificity of 99.99%were determined.

FIG. 51 shows a histogram derived from plotting the absorbance readingsversus the frequency of the values (N=45,840). In agreement with theprevious information, the combined data from the two groups indicatethat the universal cutoff value of 0.5254 lies near the midpoint betweenthe distribution of CPE-positive and CPE-negative wells. The frequencydistribution shows that the recommended cutoff value resides in theleft-most tail of the distribution of the control wells, correspondingto a probability of 0.007% in the tail extending towards the left. Thecutoff value, 0.5254, also corresponds to a tail probability of 0.02%,when cutoff values were estimated using absorbance values from allCPE-positive wells. Furthermore, the distribution profiles evident inFIG. 51 highlight that absorbance values for CPE-positive wells arewidely separated from absorbance values for CPE-negative wells

A Comparison of the Mean Absorbance Values Obtained for CPE-NegativeControl Wells Generated estimated a cutoff value of 0.5254 based onabsorbance values from 6720 control wells was previously done. A meanabsorbance value of 1.261 from the control wells was obtained with astandard deviation of 0.15. As shown in Table 57, the present studygenerated an additional 9168 control wells; 2880 were obtained from thesecond group and 6288 from the first group. Mean absorbance values of1.226 and 1.235 were obtained from the second and first groups,respectively, with an overall mean absorbance value of 1.231. Thedifference between the combined mean absorbance value and thatpreviously reported was only 0.03 absorbance values (see Table 57). Thisis a very small difference given that the data were generated over a6-month period. The studies described previously were conducted over twoconsecutive months, while the studies described herein were conducted insecond groups four months prior to that done by the first group.

Table 58 summarizes the potency values obtained for the three referencevirus strains using the different instruments in the two groups in orderto perform an instrument to instrument comparison. Six analysts from thefirst group performed the SemiAutomated TCID₅₀ Assay using two sets ofinstruments (designated AZ-039 and AZ-040). For A/New Caledonia/20/99the overall mean ranged from 9.2 to 9.3 log10TCID₅₀/mL and the titer didnot vary more than 0.09 log10TCID₅₀/mL between AZ-039 and AZ-040 (seeTable 58). For A/Sydney/05/97 the overall mean ranged from 8.5 to 8.6log10TCID₅₀/mL and did not vary more than 0.02 log10TCID₅₀/mL betweenAZ-039 and AZ-040. For B/Yamanashi/166/98 the overall mean ranged from8.3 to 8.4 log10TCID₅₀/mL and did not vary more than 0.12 log10TCID₅₀/mLbetween AZ-039 and AZ-040. The second group produced results using oneinstrument system (AZ-036). Three analysts performed the SemiAutomatedTCID₅₀ assay on three days with the three reference virus samples. Theresults of the mean difference between instruments in the second groupand Quality Control Laboratory did not vary more than 0.09log10TCID₅₀/mL for A/New Caledonia/20/99, 0.08 log10TCID₅₀/mL forA/Sydney/05/97 and 0.12 log10TCID₅₀/mL for B/Yamanashi/166/98. The meandifference data was calculated between the two instruments in the firstgroup (AZ-039 and AZ-040) and between the first and second groups(AZ-036).

In order to do an analyst to analyst comparison, in the first group eachanalyst (Analyst 1 through 6) performed a SemiAutomated TCID₅₀ potencyassay for A/New Caledonia/20/99, A/New Sydney/05/97, andB/Yamanashi/166/98 on Instrument AZ-039 over three days. In the secondgroup, each of three analysts (Analyst 7 through 9) performed theSemiAutomated TCID₅₀ potency assay with the same virus strains overthree days on Instrument AZ-036. The potency values were calculated foreach virus and are shown in Table 59. The variability among the firstgroup's results was less than or equal to 0.3 log10TCID50/mL for thethree virus samples tested. Similarly, the variability among the secondgroup's potency values was less than or equal to 0.2 log10TCID₅₀/mL. Anoverall comparison between the two groups of analysts was less than ±0.3log10TCID50/mL for the three reference virus samples. The standarddeviations (SD) for the test results (four replicates tested over threedays) ranged between 0.11 and 0.27. Because the SD values were less thanthe acceptance criterion value of 0.₅₀, all were valid.

The results provided in this section validate a “universal cutoff”absorbance value (0.5254). There is a high level of confidence in theuniversal cutoff value because the studies produce strongly concordantdata despite being generated by multiple analysts in independent assaygroups over a relatively long timeframe. To summarize, control(CPE-negative) absorbance values generated by the two groups not onlyagreed with each other, but were virtually the same as the mean valuereported previously (see, Table 57). The sensitivity and specificityvalues were very similar between the two groups; and agreed withprevious work (see FIG. 51 and Table 57). In the two groups, the“universal cutoff” for the SemiAutomated TCID₅₀ potency assay, using MTTto assess CPE, produced potency values that were comparable to eachother and to those obtained by the validated manual TCID₅₀ potencyassay. Finally, the SemiAutomated system has several proceduraladvantages over the manual method. The use of instrumentation to replacethe labor-intensive steps of manually pipetting and microscopicallyexamining assay plates increases capacity and allows for a higherthroughput. In addition, the spectrophotometric CPE readout andsubsequent automated potency calculations provide a printout and/or anelectronic record of the results.

Definitions

Unless defined otherwise, all scientific and technical terms areunderstood to have the same meaning as commonly used in the art to whichthey pertain. For the purpose of the present invention the followingterms are defined below.

The terms “nucleic acid,” “polynucleotide,” “polynucleotide sequence”and “nucleic acid sequence” refer to single-stranded or double-strandeddeoxyribonucleotide or ribonucleotide polymers, or chimeras or analoguesthereof. As used herein, the term optionally includes polymers ofanalogs of naturally occurring nucleotides having the essential natureof natural nucleotides in that they hybridize to single-stranded nucleicacids in a manner similar to naturally occurring nucleotides (e.g.,peptide nucleic acids). Unless otherwise indicated, a particular nucleicacid sequence optionally encompasses complementary sequences, inaddition to the sequence explicitly indicated.

The term “gene” is used broadly to refer to any nucleic acid associatedwith a biological function. Thus, genes include coding sequences and/orthe regulatory sequences required for their expression. The term “gene”applies to a specific genomic sequence, as well as to a cDNA or an mRNAencoded by that genomic sequence.

Genes also include non-expressed nucleic acid segments that, forexample, form recognition sequences for other proteins. Non-expressedregulatory sequences include “promoters” and “enhancers,” to whichregulatory proteins such as transcription factors bind, resulting intranscription of adjacent or nearby sequences. A “tissue specific”promoter or enhancer is one which regulates transcription in a specifictissue type or cell type, or types.

The term “vector” refers to the means by which a nucleic acid can bepropagated and/or transferred between organisms, cells, or cellularcomponents. Vectors include plasmids, viruses, bacteriophage,pro-viruses, phagemids, transposons, and artificial chromosomes, and thelike, that replicate autonomously or can integrate into a chromosome ofa host cell. A vector can also be a naked RNA polynucleotide, a nakedDNA polynucleotide, a polynucleotide composed of both DNA and RNA withinthe same strand, a poly-lysine-conjugated DNA or RNA, apeptide-conjugated DNA or RNA, a liposome-conjugated DNA, or the like,that are not autonomously replicating. Most commonly, the vectors ofherein refer to plasmids.

An “expression vector” is a vector, such as a plasmid that is capable ofpromoting expression, as well as replication of, a nucleic acidincorporated therein. Typically, the nucleic acid to be expressed is“operably linked” to a promoter and/or enhancer, and is subject totranscription regulatory control by the promoter and/or enhancer.

A “bi-directional expression vector” is characterized by two alternativepromoters oriented in the opposite direction relative to a nucleic acidsituated between the two promoters, such that expression can beinitiated in both orientations resulting in, e.g., transcription of bothplus (+) or sense strand, and negative (−) or antisense strand RNAs.

In the context of the invention, the term “isolated” refers to abiological material, such as a nucleic acid or a protein, which issubstantially free from components that normally accompany or interactwith it in its naturally occurring environment. The isolated materialoptionally comprises material not found with the material in its naturalenvironment, e.g., a cell. For example, if the material is in itsnatural environment, such as a cell, the material has been placed at alocation in the cell (e.g., genome or genetic element) not native to amaterial found in that environment. For example, a naturally occurringnucleic acid (e.g., a coding sequence, a promoter, an enhancer, etc.)becomes isolated if it is introduced by non-naturally occurring means toa locus of the genome (e.g., a vector, such as a plasmid or virusvector, or amplicon) not native to that nucleic acid. Such nucleic acidsare also referred to as “heterologous” nucleic acids.

The term “recombinant” indicates that the material (e.g., a nucleic acidor protein) has been artificially or synthetically (non-naturally)altered. The alteration can be performed on the material within, orremoved from, its natural environment or state. Specifically, whenreferring to a virus, e.g., an influenza virus, is recombinant when itis produced by the expression of a recombinant nucleic acid.

The term “reassortant,” when referring to a virus, indicates that thevirus includes genetic and/or polypeptide components derived from morethan one parental viral strain or source. For example, a 7:1 reassortantincludes 7 viral genomic segments (or gene segments) derived from afirst parental virus, and a single complementary viral genomic segment,e.g., encoding hemagglutinin or neuraminidase, from a second parentalvirus. A 6:2 reassortant includes 6 genomic segments, most commonly the6 internal genes from a first parental virus, and two complementarysegments, e.g., hemagglutinin and neuraminidase, from a differentparental virus.

The term “introduced” when referring to a heterologous or isolatednucleic acid refers to the incorporation of a nucleic acid into aeukaryotic or prokaryotic cell where the nucleic acid can beincorporated into the genome of the cell (e.g., chromosome, plasmid,plastid or mitochondrial DNA), converted into an autonomous replicon, ortransiently expressed (e.g., transfected mRNA). The term includes suchmethods as “infection,” “transfection,” “transformation,” and“transduction.” In the context of the invention, a variety of methodscan be employed to introduce nucleic acids into prokaryotic cells,including electroporation, calcium phosphate precipitation, lipidmediated transfection (lipofection), etc.

The term “host cell” means a cell that contains a heterologous nucleicacid, such as a vector, and supports the replication and/or expressionof the nucleic acid. Host cells can be prokaryotic cells such as E.coli, or eukaryotic cells such as yeast, insect, amphibian, avian ormammalian cells, including human cells. Exemplary host cells in thecontext of the invention include Vero (African green monkey kidney)cells, BHK (baby hamster kidney) cells, primary chick kidney (PCK)cells, Madin-Darby Canine Kidney (MDCK) cells, Madin-Darby Bovine Kidney(MDBK) cells, 293 cells (e.g., 293T cells), and COS cells (e.g., COS1,COS7 cells).

Influenza Virus

The compositions and methods herein are primarily concerned withproduction of influenza viruses for vaccines. Influenza viruses are madeup of an internal ribonucleoprotein core containing a segmentedsingle-stranded RNA genome and an outer lipoprotein envelope lined by amatrix protein. Influenza A and influenza B viruses each contain eightsegments of single stranded negative sense RNA. The influenza A genomeencodes eleven polypeptides. Segments 1–3 encode three polypeptides,making up a RNA-dependent RNA polymerase. Segment 1 encodes thepolymerase complex protein PB2. The remaining polymerase proteins PB1and PA are encoded by segment 2 and segment 3, respectively. Inaddition, segment 1 of some influenza strains encodes a small protein,PB1-F2, produced from an alternative reading frame within the PB 1coding region. Segment 4 encodes the hemagglutinin (HA) surfaceglycoprotein involved in cell attachment and entry during infection.Segment 5 encodes the nucleocapsid nucleoprotein (NP) polypeptide, themajor structural component associated with viral RNA. Segment 6 encodesa neuraminidase (NA) envelope glycoprotein. Segment 7 encodes two matrixproteins, designated M1 and M2, which are translated from differentiallyspliced mRNAs. Segment 8 encodes NS1 and NS2, two nonstructuralproteins, which are translated from alternatively spliced mRNA variants.

The eight genome segments of influenza B encode 11 proteins. The threelargest genes code for components of the RNA polymerase, PB1, PB2 andPA. Segment 4 encodes the HA protein. Segment 5 encodes NP. Segment 6encodes the NA protein and the NB protein. Both proteins, NB and NA, aretranslated from overlapping reading frames of a biscistronic mRNA.Segment 7 of influenza B also encodes two proteins: M1 and BM2. Thesmallest segment encodes two products, NS1 which is translated from thefull length RNA, and NS2 which is translated from a spliced mRNAvariant.

Influenza Virus Vaccine

Historically, influenza virus vaccines have primarily been produced inembryonated hen eggs using strains of virus selected based on empiricalpredictions of relevant strains. More recently, reassortant viruses havebeen produced that incorporate selected hemagglutinin and neuraminidaseantigens in the context of an approved attenuated, temperature sensitivemaster strain. Following culture of the virus through multiple passagesin hen eggs, influenza viruses are recovered and, optionally,inactivated, e.g., using formaldehyde and/or β-propiolactone (oralternatively used in live attenuated vaccines).

However, production of influenza vaccine in this manner has severalsignificant concerns. For example, contaminants remaining from the heneggs can be highly antigenic and/or pyrogenic, and can frequently resultin significant side effects upon administration. Thus, as describedherein, one aspect of the current invention involves replacement of somepercentage of egg components with animal free media. More importantly,virus strains designated for vaccine production must be selected anddistributed, typically months in advance of the next flu season to allowtime for production and inactivation of influenza vaccine. Again, anyimprovements in production time, e.g., as through use of the methods andcompositions of the current invention, are thus quite desirable.

Attempts at producing recombinant and reassortant vaccines in cellculture have been hampered by the inability of some of the strainsapproved for vaccine production to grow efficiently under standard cellculture conditions. Thus, prior work by the inventors and theircoworkers provided a vector system, and methods for producingrecombinant and reassortant viruses in culture, thus, making it possibleto rapidly produce vaccines corresponding to one or many selectedantigenic strains of virus. See, Multi-Plasmid System for the productionof Influenza virus, cited above. Of course, such reassortments areoptionally further amplified in hen eggs. Typically, the cultures aremaintained in a system, such as a cell culture incubator, undercontrolled humidity and CO₂, at constant temperature using a temperatureregulator, such as a thermostat to insure that the temperature does notexceed 35 ° C. Such pioneering work, as well as other vaccineproduction, can be further optimized and streamlined through use of thecurrent invention in whole or part.

Reassortant influenza viruses can be readily obtained by introducing asubset of vectors corresponding to genomic segments of a masterinfluenza virus, in combination with complementary segments derived fromstrains of interest (e.g., antigenic variants of interest). Typically,the master strains are selected on the basis of desirable propertiesrelevant to vaccine administration. For example, for vaccine production,e.g., for production of a live attenuated vaccine, the master donorvirus strain may be selected for an attenuated phenotype, coldadaptation and/or temperature sensitivity.

FluMist™

As mentioned previously, numerous examples and types of influenzavaccine exist. An exemplary influenza vaccine is FluMist™ which is alive, attenuated vaccine that protects children and adults frominfluenza illness (Belshe et al. (1998) The efficacy of live attenuated,cold-adapted, trivalent, intranasal influenza virus vaccine in childrenN. Engl. J. Med. 338:1405–12; Nichol et al. (1999) Effectiveness oflive, attenuated intranasal influenza virus vaccine in healthy, workingadults: a randomized controlled trial JAMA 282:137–44). In typicalembodiments, the methods and compositions of the current invention arepreferably adapted to, or used with, production of FluMist™ vaccine.However, it will be appreciated by those skilled in the art that thesteps/compositions herein are also adaptable to production of similar oreven different viral vaccines.

FluMist™ vaccine strains contain, e.g., HA and NA gene segments derivedfrom the wild-type strains to which the vaccine is addressed along withsix gene segments, PB1, PB2, PA, NP, M and NS, from a common masterdonor virus (MDV). The MDV for influenza A strains of FluMist (MDV-A),was created by serial passage of the wild-type A/Ann Arbor/6/60(A/AA/6160) strain in primary chicken kidney tissue culture atsuccessively lower temperatures (Maassab (1967) Adaptation and growthcharacteristics of influenza virus at 25 degrees C. Nature 213:612–4).MDV-A replicates efficiently at 25° C. (ca, cold adapted), but itsgrowth is restricted at 38 and 39° C. (ts, temperature sensitive).Additionally, this virus does not replicate in the lungs of infectedferrets (att, attenuation). The ts phenotype is believed to contributeto the attenuation of the vaccine in humans by restricting itsreplication in all but the coolest regions of the respiratory tract. Thestability of this property has been demonstrated in animal models andclinical studies. In contrast to the ts phenotype of influenza strainscreated by chemical mutagenesis, the ts property of MDV-A does notrevert following passage through infected hamsters or in shed isolatesfrom children (for a recent review, see Murphy & Coelingh (2002)Principles underlying the development and use of live attenuatedcold-adapted influenza A and B virus vaccines Viral Immunol.15:295–323).

Clinical studies in over 20,000 adults and children involving 12separate 6:2 reassortant strains have shown that these vaccines areattenuated, safe and efficacious (Belshe et al. (1998) The efficacy oflive attenuated, cold-adapted, trivalent, intranasal influenza virusvaccine in children N. Engl. J. Med. 338:1405–12; Boyce et al. (2000)Safety and immunogenicity of adjuvanted and unadjuvanted subunitinfluenza vaccines administered intranasally to healthy adults Vaccine19:217–26; Edwards et al. (1994) A randomized controlled trial of coldadapted and inactivated vaccines for the prevention of influenza Adisease J. Infect. Dis. 169:68–76 ; Nichol et al. (1999) Effectivenessof live, attenuated intranasal influenza virus vaccine in healthy,working adults: a randomized controlled trial JAMA 282:137–44).Reassortants carrying the six internal genes of MDV-A and the two HA andNA gene segments of a wild-type virus (i.e., a 6:2 reassortant)consistently maintain ca, ts and att phenotypes (Maassab et al. (1982)Evaluation of a cold-recombinant influenza virus vaccine in ferrets J.Infect. Dis. 146:780–900). Production of such reassorted virus using Bstrains of influenza are is more difficult, however.

Recent work, see, Multi-Plasmid System for the Production of InfluenzaVirus, cited above, has shown an eight plasmid system for the generationof influenza B virus entirely from cloned cDNA, and methods for theproduction of attenuated live influenza A and B virus suitable forvaccine formulations, such as live virus vaccine formulations useful forintranasal administration.

The system and methods described previously are useful for the rapidproduction in cell culture of recombinant and reassortant influenza Aand B viruses, including viruses suitable for use as vaccines, includinglive attenuated vaccines, such as vaccines suitable for intranasaladministration such as FluMist®. The methods of the current inventionherein, are optionally used in conjunction with or in combination withsuch previous work involving, e.g., reassorted influenza viruses forvaccine production to produce viruses for vaccines in a more stable,consistent and productive manner.

Cell Culture

As previously stated, influenza virus optionally can be grown in cellculture. Typically, propagation of the virus is accomplished in themedia compositions in which the host cell is commonly cultured. Suitablehost cells for the replication of influenza virus include, e.g., Verocells, BHK cells, MDCK cells, 293 cells and COS cells, including 293Tcells, COS7 cells. Commonly, co-cultures including two of the above celllines, e.g., MDCK cells and either 293T or COS cells are employed at aratio, e.g., of 1:1, to improve replication efficiency. Typically, cellsare cultured in a standard commercial culture medium, such as Dulbecco'smodified Eagle's medium supplemented with serum (e.g., 10% fetal bovineserum), or in serum free medium, under controlled humidity and CO₂concentration suitable for maintaining neutral buffered pH (e.g., at pHbetween 7.0 and 7.2). Optionally, the medium contains antibiotics toprevent bacterial growth, e.g., penicillin, streptomycin, etc., and/oradditional nutrients, such as L-glutamine, sodium pyruvate,non-essential amino acids, additional supplements to promote favorablegrowth characteristics, e.g., trypsin, β-mercaptoethanol, and the like.

Procedures for maintaining mammalian cells in culture have beenextensively reported, and are well known to those of skill in the art.General protocols are provided, e.g., in Freshney (1983) Culture ofAnimal Cells: Manual of Basic Technique, Alan R. Liss, New York; Paul(1975) Cell and Tissue Culture, 5^(th) ed., Livingston, Edinburgh; Adams(1980) Laboratory Techniques in Biochemistry and Molecular Biology-CellCulture for Biochemists, Work and Burdon (eds.) Elsevier, Amsterdam.Additional details regarding tissue culture procedures of particularinterest in the production of influenza virus in vitro include, e.g.,Merten et al. (1996) Production of influenza virus in cell cultures forvaccine preparation in Cohen and Shafferman (eds.) Novel Strategies inDesign and Production of Vaccines, which is incorporated herein in itsentirety for all purposes. Additionally, variations in such proceduresadapted to the present invention are readily determined through routineexperimentation and will be familiar to those skilled in the art.

Cells for production of influenza virus can be cultured inserum-containing or serum free medium. In some case, e.g., for thepreparation of purified viruses, it is typically desirable to grow thehost cells in serum free conditions. Cells can be cultured in smallscale, e.g., less than 25 ml medium, culture tubes or flasks or in largeflasks with agitation, in rotator bottles, or on microcarrier beads(e.g., DEAE-Dextran microcarrier beads, such as Dormacell, Pfeifer &Langen; Superbead, Flow Laboratories; styrene copolymer-tri-methylaminebeads, such as Hillex, SoloHill, Ann Arbor) in flasks, bottles orreactor cultures. Microcarrier beads are small spheres (in the range of100–200 microns in diameter) that provide a large surface area foradherent cell growth per volume of cell culture. For example a singleliter of medium can include more than 20 million microcarrier beadsproviding greater than 8000 square centimeters of growth surface. Forcommercial production of viruses, e.g., for vaccine production, it isoften desirable to culture the cells in a bioreactor or fermenter.Bioreactors are available in volumes from under 1 liter to in excess of100 liters, e.g., Cyto3 Bioreactor (Osmonics, Minnetonka, Minn.); NBSbioreactors (New Brunswick Scientific, Edison, N.J.); laboratory andcommercial scale bioreactors from B. Braun Biotech International (B.Braun Biotech, Melsungen, Germany).

Regardless of the culture volume, in many desired aspects of the currentinvention, it is important that the cultures be maintained at anappropriate temperature, to insure efficient recovery of recombinantand/or reassortant influenza virus using temperature dependent multiplasmid systems (see, e.g., Multi-Plasmid System for the Production ofInfluenza Virus, cited above), heating of virus solutions forfiltration, etc. Typically, a regulator, e.g., a thermostat, or otherdevice for sensing and maintaining the temperature of the cell culturesystem and/or other solution, is employed to insure that the temperatureis at the correct level during the appropriate period (e.g., virusreplication, etc.).

In some embodiments herein (e.g., wherein reasserted viruses are to beproduced from segments on vectors) vectors comprising influenza genomesegments are introduced (e.g., transfected) into host cells according tomethods well known in the art for introducing heterologous nucleic acidsinto eukaryotic cells, including, e.g., calcium phosphateco-precipitation, electroporation, microinjection, lipofection, andtransfection employing polyamine transfection reagents. For example,vectors, e.g., plasmids, can be transfected into host cells, such as COScells, 293T cells or combinations of COS or 293T cells and MDCK cells,using the polyamine transfection reagent TransIT-LT1 (Mirus) accordingto the manufacturer's instructions in order to produce reassertedviruses, etc. Approximately 1 μg of each vector to be introduced intothe population of host cells with approximately 2 μl of TransIT-LT1diluted in 160 μl medium, preferably serum-free medium, in a totalvolume of 200 μl. The DNA:transfection reagent mixtures are incubated atroom temperature for 45 minutes followed by addition of 800 μl ofmedium. The transfection mixture is added to the host cells, and thecells are cultured as described above or via other methods well known tothose skilled in the art. Accordingly, for the production of recombinantor reassortant viruses in cell culture, vectors incorporating each ofthe 8 genome segments, (PB2, PB1, PA, NP, M, NS, HA and NA) are mixedwith approximately 20 μl TransIT-LT1 and transfected into host cells.Optionally, serum-containing medium is replaced prior to transfectionwith serum-free medium, e.g., Opti-MEM I, and incubated for 4–6 hours.

Alternatively, electroporation can be employed to introduce such vectorsincorporating influenza genome segments into host cells. For example,plasmid vectors incorporating an influenza A or influenza B virus arefavorably introduced into Vero cells using electroporation according tothe following procedure. In brief, approximately 5×10⁶ Vero cells, e.g.,grown in Modified Eagle's Medium (MEM) supplemented with 10% FetalBovine Serum (FBS) are resuspended in 0.4 ml OptiMEM and placed in anelectroporation cuvette. Twenty micrograms of DNA in a volume of up to25 μl is added to the cells in the cuvette, which is then mixed gentlyby tapping. Electroporation is performed according to the manufacturer'sinstructions (e.g., BioRad Gene Pulser II with Capacitance Extender Plusconnected) at 300 volts, 950 microFarads with a time constant of between28–33 msec. The cells are remixed by gently tapping and, approximately1–2 minutes following electroporation, 0.7 ml MEM with 10% FBS is addeddirectly to the cuvette. The cells are then transferred to two wells ofa standard 6 well tissue culture dish containing 2 ml MEM, 10% FBS. Thecuvette is washed to recover any remaining cells and the wash suspensionis divided between the two wells. Final volume is approximately 3.5 mL.The cells are then incubated under conditions permissive for viralgrowth, e.g., at approximately 33° C. for cold adapted strains.

Kits

To facilitate use of the methods and compositions of the invention, anyof the vaccine components and/or compositions, e.g., reasserted virus inallantoic fluid, and various formulations, etc., and additionalcomponents, such as, buffer, cells, culture medium, useful for packagingand infection of influenza viruses for experimental or therapeuticvaccine purposes, can be packaged in the form of a kit. Typically, thekit contains, in addition to the above components, additional materialswhich can include, e.g., instructions for performing the methods of theinvention, packaging material, and a container.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques and apparatus described abovemay be used in various combinations. All publications, patents, patentapplications, or other documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, patent, patent application, orother document were individually indicated to be incorporated byreference for all purposes.

TABLE 1 Description of Step Detail of Step Step 1. Co-infection ofmaster donor virus (MDV) and WT virus in CEK cells. Step 2. Selection ofreassorted viruses. Depending on virus strain, can be done in eggs or inCEK cells. Select for MDV NA and/or HA. Step 3. Cloning of reassortedviruses. Step 4. Purification of reassorted viruses in eggs. Step 5.Expansion of reassorted viruses in eggs to generate a master viralstrain (MVS). Step 6. Expansion of MVS to produce a master working viralstrain (MWVS). Step 7. Conditioning, washing, primary Eggs containingthe incubation of the eggs, and reassorted virus are inoculation.optionally rocked during incubation. Step 8. Candling, inoculation,sealing, secondary incubation, etc., of eggs. Step 9. Candling of theeggs and chilling. Step 10. Harvesting of virus solution from the Viruscontaining eggs. solutions are optionally warmed and sterile filtered toremove impurities/contaminants (bioburden). Step 11. Clarification ofthe virus solution. The solution is also optionally ultrafiltered to,e.g., remove uric acid and other animal derived impurities and tostabilize the solution. Step 12. Stabilization of the virus solution.Arginine is optionally added either in addition to or in place ofgelatin or gelatin hydrolysate at pH 6.6 to 8.0 to stabilize thesolution. Use of arginine exclusively avoids the introduction ofadditional animal products. Step 13. Potency assay of the virussolutions. Optional use of a “universal reagent” and field focus assaysas opposed to, e.g., TCID50 to determine potency. Step 14. Sterilityassay of the virus solutions. Step 15. NAF adjustment of the virus NAFis optionally solutions. reduced/ replaced with buffer, e.g., toincrease stability.

TABLE 2 Tube/Well Target of cell MOI of MOI of incubation culture MDVwild-type time in hours 1 5.0 1.0 24 2 5.0 0.2 24 3 1.0 1.0 24 4 1.0 0.224 5 1.0 0.04 24 6 n/a n/a 24

TABLE 3 Manufacture Potential Process Detection Type/Assay TimeAlternatives Egg pre/post Egg Candling Manual/hours Automated candlinginoculation of eggs or thermal imaging of eggs Virus harvest MPAManual/14 days Bioluminence based Bioburden Manual/3 days detection orMPN Virus Harvest Mycoplasma Manual/28 days PCR growth Virus harvestMycobacterium Manual/56 days PCR or clinical growth diagnostic systems

TABLE 4 Virus Type Strain and Isolate Number A H1N1 ca A/Beijing/262/95A H1N1 ca A/New Caledonia/20/99 A H3N2 ca A/Sydney/05/97 A H3N2 caA/Panama/2007/99 B ca B/Victoria/504/2000 B ca B/Yamanashi/166/98

TABLE 5 A/Sydney/05/97 Virus potency [log₁₀ TCID₅₀/mL]. TemperatureProcess step 5 ± 3° C. 20 ± 3° C. 31 ± 3° C. Stabilized VAF (beforetreatment) 8.7 ± 0.3 8.6 ± 0.2 8.8 ± 0.2 Stabilized VAF (aftertreatment) 9.0 ± 0.2 8.8 ± 0.2 8.8 ± 0.2 Filtered VAF (pool) 7.6 ± 0.27.7 ± 0.1 8.7 ± 0.1 Centrifuged Stabilized VAF (control) 8.4 ± 0.3 8.6 ±0.2 8.7 ± 0.2 Gain/Loss Filtered vs. Control −0.8 −0.7 0.0 NA = notassayed All filtrations in Table 5 were performed from the same dayharvest. Prior to filtration through Sartoclean CA and Sartopore 2filters VAF was exposed for 60 minutes to 5 ± 3° C., 20 ± 3° C. and 31 ±3° C.

TABLE 6 A/Sydney/05/97 neuraminidase activity [μU/mL]. TemperatureProcess step 5 ± 3° C. 20 ± 3° C. 31 ± 3° C. Stabilized VAF (beforetreatment) 34.4 36.4 38.4 Stabilized VAF (after treatment) 43.7 38.739.1 Filtered VAF (pool) BD BD 22.1 Centrifuged Stabilized VAF (control)28.2 27.5 27.0 Gain/Loss Filtered vs. Control −28.2 −27.5 −4.9 BD =below detection (less than 5 μU/mL) All filtrations in Table 6 wereperformed from the same day harvest. Prior to filtration throughSartoclean CA and Sartopore 2 filters VAF was exposed for 60 minutes to5 ± 3° C., 20 ± 3° C. and 31 ± 3° C.

TABLE 7 A/Sydney/05/97 hemagglutinin activity [HA titer]. TemperatureProcess step 5 ± 3° C. 20 ± 3° C. 31 ± 3° C. Stabilized VAF (beforetreatment) 128 128 256 Stabilized VAF (after treatment) 128 256 128Filtered VAF (pool) 4 16 64 Centrifuged Stabilized VAF (control) 128 64256 All filtrations in Table 7 were performed from the same day harvest.Prior to filtration through Sartoclean CA and Sartopore 2 filters VAFwas exposed for 60 minutes to 5 ± 3° C., 20 ± 3° C. and 31 ± 3° C.

TABLE 8 A/Sydney/05/97 Virus potency [log₁₀ TCID₅₀/mL]. TemperatureProcess step 5 ± 3° C. 20 ± 3° C. 31 ± 3° C. Stabilized VAF (beforetreatment) 8.7 ± 0.1 8.5 ± 0.2 8.8 ± 0.1 Stabilized VAF (aftertreatment) 8.9 ± 0.2 8.9 ± 0.2 8.7 ± 0.2 Filtered VAF (pool) 7.6 ± 0.27.5 ± 0.2 8.7 ± 0.2 Centrifuged Stabilized VAF (control) 8.5 ± 0.2 8.5 ±0.1 8.7 ± 0.1 Gain/Loss Filtered vs. Control −0.9 −1.0 0.0 Allfiltrations in Table 8 were performed from the same day harvest. Priorto filtration through Sartoclean CA and Sartopore 2 filters VAF wasexposed for 60 minutes to 5 ± 3° C., 20 ± 3° C. and 31 ± 3° C.*.

TABLE 9 A/Sydney/05/97 neuraminidase activity [μU/mL]. TemperatureProcess step 5 ± 3° C. 20 ± 3° C. 31 ± 3° C. Stabilized VAF (beforetreatment) 29.8 26.1 27.0 Stabilized VAF (after treatment) 29.3 26.127.3 Filtered VAF (pool) BD BD 15.4 Centrifuged Stabilized VAF (control)21.3 16.1 20.3 Gain/Loss Filtered vs. Control −21.3 −16.1 −4.9 BD =below detection (less than 5 μU/mL) All filtrations were performed fromthe same day harvest. Prior to filtration through Sartoclean CA andSartopore 2 filters VAF was exposed for 60 minutes to 5 ± 3° C., 20 ± 3°C. and 31 ± 3° C.*.

TABLE 10 A/Sydney/05/97 hemagglutinin activity [HA titer]. TemperatureProcess step 5 ± 3° C. 20 ± 3° C. 31 ± 3° C. Stabilized VAF (beforetreatment) 256 128 256 Stabilized VAF (after treatment) 256 256 128Filtered VAF (pool) 16 32 128 Centrifuged Stabilized VAF (control) 128128 128 *10% of PBS was added to all experiments to adjust volume Allfiltrations in Table 10 were performed from the same day harvest. Priorto filtration through Sartoclean CA and Sartopore 2 filters VAF wasexposed for 60 minutes to 5 ± 3° C., 20 ± 3° C. and 31 ± 3° C.*.

TABLE 11 A/Sydney/05/97virus potency [log₁₀ TCID₅₀/mL]. Warming timeProcess step 30 min 90 min 180 min Stabilized VAF (before warming)* 8.7± 0.2 8.7 ± 0.2 8.7 ± 0.2 Stabilized VAF (warmed up) 8.9 ± 0.2 8.9 ± 0.28.7 ± 0.2 Filtered VAF (pool) 8.5 ± 0.2 8.5 ± 0.2 8.9 ± 0.3 CentrifugedStabilized VAF 8.9 ± 0.3 8.9 ± 0.3 8.9 ± 0.3 (control)* Gain/LossFiltered vs. Control −0.4 −0.4 0.0 All filtrations were performed fromthe same day harvest. Prior to filtration through Sartoclean CA andSartopore 2 filters VAF was exposed to 31 ± 3° C. for 30, 90 or 180minutes.

TABLE 12 A/Sydney/05/97 neuraminidase activity [μU/mL]. Warming timeProcess step 30 min 90 min 180 min Stabilized VAF (before warming)* 35.035.0 35.0 Stabilized VAF (warmed up) 38.0 35.2 36.0 Filtered VAF (pool)17.8 22.9 23.1 Centrifuged Stabilized VAF (control)* 27.9 27.9 27.9Gain/loss Filtered vs. Control −10.1 −5.0 −4.8 All filtrations wereperformed from the same day harvest. Prior to filtration throughSartoclean CA and Sartopore 2 filters VAF was exposed to 31 ± 3° C. for30, 90 or 180 minutes.

TABLE 13 A/Sydney/05/97 hemagglutinin activity [HA titer]. Warming timeProcess step 30 min 90 min 180 min Stabilized VAF (before warming)* 256256 256 Stabilized VAF (warmed up) 128 256 256 Filtered VAF (pool) 128128 128 Centrifuged Stabilized VAF (control)* NA NA NA *Stabilized VAFand Centrifuged Stabilized VAF (control) samples were taken from thepool before VAF was divided into 4 individual experiments (0, 30, 60 or90 minutes temperature treatment). NA = not available All filtrationswere performed from the same day harvest. Prior to filtration throughSartoclean CA and Sartopore 2 filters, VAF was exposed to 31 ± 3° C. for30, 90 or 180 minutes.

TABLE 14 A/Sydney/05/97virus potency [log₁₀ TCID₅₀/mL]. Warming timeProcess step 0 min 30 min 60 min 90 min Stabilized VAF (before warming)*8.8 ± 0.3 8.8 ± 0.3 8.8 ± 0.3 8.8 ± 0.3 Stabilized VAF (warmed up) — 8.7± 0.2 8.6 ± 0.1 8.6 ± 0.1 Filtered VAF (pool) 7.7 ± 0.1 8.3 ± 0.2 8.4 ±0.2 8.6 ± 0.1 Centrifuged Stabilized VAF (control)* 8.6 ± 0.1 8.6 ± 0.18.6 ± 0.1 8.6 ± 0.1 Gain/loss Filtered vs. Control −0.9 −0.3 −0.2 0.0All filtrations were performed from the same day harvest. Prior tofiltration through Sartoclean CA and Sartopore 2 filters, VAF wasexposed to 31 ± 3° C. for 0, 30, 60 or 90 minutes.

TABLE 15 A/Sydney/05/97 neuraminidase activity [μU/mL]. Warming timeProcess step 0 min 30 min 60 min 90 min Stabilized VAF (before warming)*44.5 44.5 44.5 44.5 Stabilized VAF (warmed up) — 44.5 41.0 47.5 FilteredVAF (pool) 6.0 17.5 26.0 30.0 Centrifuged Stabilized VAF 33.0 33.0 33.033.0 (control)* Gain/loss Filtered vs. Control −27.0 −15.5 −7.0 −3.0 Allfiltrations were performed from the same day harvest. Prior tofiltration through Sartoclean CA and Sartopore 2 filters, VAF wasexposed to 31 ± 3° C. for 0, 30, 60 or 90 minutes.

TABLE 16 A/Sydney/05/97 hemagglutinin activity [HA titer]. Warming timeProcess step 0 min 30 min 60 min 90 min Stabilized VAF (before warming)*64 64 64 64 Stabilized VAF (warmed up) — 128 128 128 Filtered VAF (pool)16 64 128 128 Centrifuged Stabilized VAF 64 64 64 64 (control)**Stabilized VAF and Centrifuged Stabilized VAF (control) samples weretaken from the pool before VAF was divided into 4 individual experiments(0, 30, 60 or 90 minutes temperature treatment). All filtrations wereperformed from the same day harvest. Prior to filtration throughSartoclean CA and Sartopore 2 filters, VAF was exposed to 31 ± 3° C. for0, 30, 60 or 90 minutes.

TABLE 17 Virus potency [log₁₀ TCID₅₀/mL]. Warming time Process step 0min 30 min 60 min 90 min Stabilized VAF (before warming)* 8.6 ± 0.2 8.6± 0.2 8.6 ± 0.2 8.6 ± 0.2 Stabilized VAF (warmed up) — 8.6 ± 0.2 8.6 ±0.2 8.5 ± 0.3 Filtered VAF (pool) 8.1 ± 0.2 8.1 ± 0.2 8.5 ± 0.2 8.5 ±0.3 Centrifuged Stabilized VAF (control)* 8.6 ± 0.2 8.6 ± 0.2 8.6 ± 0.28.6 ± 0.2 Gain/Loss Filtered vs. Control −0.5 −0.5 −0.1 −0.1 Allfiltrations were performed from the same day harvest. Prior tofiltration through Sartoclean CA and Sartopore 2 filters, VAF wasexposed to 31 ± 3° C. for 0 30, 60 or 90 minutes.

TABLE 18 Neuraminidase activity μU/mL. Warming time Process step 0 min30 min 60 min 90 min Stabilized VAF (before warming)* 35.5 35.5 35.535.5 Stabilized VAF (warmed up) — 36.5 36.0 34.5 Filtered VAF (pool) 7.09.0 14.5 19.5 Centrifuged Stabilized VAF 23.0 23.0 23.0 23.0 (control)*Gain/Loss Filtered vs. Control −16 −14 −8.5 −3.5 All filtrations wereperformed from the same day harvest. Before filtration throughSartoclean CA Sartopore 2 filters, VAF was exposed to 31 ± 3° C. for 030, 60 or 90 minutes.

TABLE 19 Hemagglutinin activity. Warming time Process step 0 min 30 min60 min 90 min Stabilized VAF (before warming)* 64 64 64 64 StabilizedVAF (warmed up) — 64 64 128 Filtered VAF (pool) 16 32 64 64 CentrifugedStabilized VAF 64 64 64 64 (control)* *Stabilized VAF and CentrifugedStabilized VAF (control) samples were taken from the pool before VAF wasdivided into 4 individual experiments (0, 30, 60 or 90 minutestemperature treatment). All filtrations were performed from the same dayharvest. Before filtration through Sartoclean CA Sartopore2 filters VAFwas exposed to 31 ± 3° C. for 0 30, 60 or 90 minutes.

TABLE 20 Virus potency [log₁₀ TCID₅₀/mL] of six influenza strains.Process step Centrifuged Stabilized Filtered Stabilized PotencyInfluenza strain VAF* VAF VAF* Gain/Loss A/Beijing/262/95 RT** 9.7 ± 0.19.6 ± 0.2 9.6 ± 0.3 0.0 A/Beijing/262/95 31 ± 3° C. 9.7 ± 0.1 9.4 ± 0.39.6 ± 0.3 −0.2 A/New Caledonia/20/99 RT** 9.6 ± 0.2 9.3 ± 0.2 9.5 ± 0.2−0.2 A/New Caledonia/20/99 31 ± 3° C. 9.6 ± 0.2 9.3 ± 0.2 9.5 ± 0.2 −0.2A/Sydney/05/97 RT 8.8 ± 0.3 7.7 ± 0.1 8.6 ± 0.1 −0.9 A/Sydney/05/97 31 ±3° C. 8.8 ± 0.3 8.4 ± 0.2 8.6 ± 0.1 −0.2 A/Panama/2007/99 RT** 8.5 ± 0.28.2 ± 0.1 8.5 ± 0.3 −0.3 A/Panama/2007/99 31 ± 3° C. 8.5 ± 0.2 8.6 ± 0.28.5 ± 0.3 0.1 B/Victoria/504/2000 RT** 8.3 ± 0.2 7.8 ± 0.2 8.4 ± 0.2−0.6 B/Victoria/504/2000 31 ± 3° C. 8.3 ± 0.2 8.4 ± 0.2 8.4 ± 0.2 0.0B/Yamanashi/166/98 RT** 8.4 ± 0.2 8.3 ± 0.2 8.6 ± 0.2 −0.3B/Yamanashi/166/98 31 ± 3° C. 8.4 ± 0.2 8.4 ± 0.2 8.6 ± 0.2 −0.2*Stabilized VAF and Centrifuged Stabilized VAF (control) samples weretaken from the pool before VAF was divided into individual experiments(RT and 31 ± 3° C.). **RT room temperature Both filtrations for the samestrain were performed from the same day harvest. Prior to filtrationthrough Sartoclean CA and Sartopore 2 filters, VAF was exposed to 31 ±3° C. for 0 (RT) or 60 minutes.

TABLE 21 Neuraminidase activity [μU/mL] of six influenza strains.Process step Centrifuged Stabilized Filtered Stabilized Influenza trainVAF VAF VAF Activity Gain/Loss A/Beijing/262/95 RT** 55.5 47.5 52.0 −4.5A/Beijing/262/95 31 ± 3° C. 55.5 51.5 52.0 −0.5 A/New Caledonia/20/99RT** 49.5 47.5 48.5 −1.0 A/New Caledonia/20/99 31 ± 3° C. 49.5 48.5 48.50.0 A/Sydney/05/97 RT** 44.5 6.0 33.0 −27.0 A/Sydney/05/97 31 ± 3° C.44.5 26.0 33.0 −7.0 A/Panama/2007/99 RT** 61.0 16.5 48.0 −31.5A/Panama/2007/99 31 ± 3° C. 61.0 40.0 48.0 −8.0 B/Victoria/504/2000 RT**58.5 20.5 44.0 −23.5 B/Victoria/504/2000 31 ± 3° C. 58.5 37.0 44.0 −7.0B/Yamanashi/166/98 RT** 66.5 51.0 55.5 −4.5 B/Yamanashi/166/98 31 ± 3°C. 66.5 53.0 55.5 −2.5 *Stabilized VAF and Centrifuged Stabilized VAF(control) samples were taken from the pool before VAF was divided intoindividual experiments (RT and 31 ± 3° C.). **RT = room temperature Bothfiltrations for the same strain were performed from the same dayharvest. Prior to filtration through Sartoclean and CA Sartopore 2filters, VAF was exposed to 31 ± 3° C. for 0 (RT) or 60 minutes.

TABLE 22 Hemagglutinin activity [HA titer] of six influenza strains.Process step Stabilized Stabilized Warmed up Filtered CentrifugedInfluenza train VAF* VAF VAF Stabilized VAF* A/Beijing/262/95 RT** 1024— 128 1024 A/Beijing/262/95 31 ± 3° C. 1024 512  512 1024 A/NewCaledonia/20/99 RT** 32 — 32 64 A/New Caledonia/20/99 31 ± 3° C. 32 3232 64 A/Sydney/05/97 RT** 64 — 16 64 A/Sydney/05/97 31 ± 3° C. 64 128 128 64 A/Panama/2007/99 RT** 128 — 32 128 A/Panama/2007/99 31 ± 3° C.128 128  64 128 B/Victoria/504/2000 RT** 128 — 32 128B/Victoria/504/2000 31 ± 3° C. 128 64 64 128 B/Yamanashi/166/98 RT** 512— 16 32 B/Yamanashi/166/98 31 ± 3° C. 512 32 32 32 *Stabilized VAF andCentrifuged Stabilized VAF (control) samples were taken from the poolbefore VAF was divided into individual experiments (RT and 31 ± 3° C.).**RT = room temperature Both filtrations for the same strain wereperformed from the same day harvest. Prior to filtration throughSartoclean and CA Sartopore 2 filters, VAF was exposed to 31 ± 3° C. for0 (RT) or 60 minutes.

TABLE 23 Virus potency [log₁₀TCID₅₀/mL] of six influenza strains.Process step Stabilized Filtered Centrifuged Potency Influenza strainVAF* VAF Stabilized VAF* Gain/Loss A/Beijing/262/95 RT** 9.6 ± 0.1 9.4 ±0.2 9.6 ± 0.1 −0.2 A/Beijing/262/95 31 ± 3° C. 9.6 ± 0.1 9.5 ± 0.2 9.6 ±0.1 −0.1 A/New Caledonia/20/99 RT** 9.1 ± 0.2 9.5 ± 0.2 9.2 ± 0.2 0.3A/New Caledonia/20/99 31 ± 3° C. 9.1 ± 0.2 9.2 ± 0.3 9.2 ± 0.2 0.0A/Sydney/05/97 RT** 8.6 ± 0.2 8.1 ± 0.2 8.6 ± 0.2 −0.5 A/Sydney/05/97 31± 3° C. 8.6 ± 0.2 8.5 ± 0.2 8.6 ± 0.2 −0.1 A/Panama/2007/99 RT** 8.9 ±0.2 8.3 ± 0.2 8.5 ± 0.2 −0.2 A/Panama/2007/99 31 ± 3° C. 8.9 ± 0.2 8.6 ±0.1 8.5 ± 0.2 0.1 B/Victoria/504/2000 RT** 7.6 ± 0.2 7.7 ± 0.2 7.9 ± 0.2−0.2 B/Victoria/504/2000 31 ± 3° C. 7.6 ± 0.2 7.7 ± 0.1 7.9 ± 0.2 −0.2B/Yamanashi/166/98 RT** 8.4 ± 0.1 8.2 ± 0.2 8.3 ± 0.3 −0.1B/Yamanashi/166/98 31 ± 3° C. 8.4 ± 0.1 8.3 ± 0.2 8.3 ± 0.3 0.0*Stabilized VAF and Centrifuged Stabilized VAF (control) samples weretaken from the pool before VAF was divided into individual experiments(RT and 31 ± 3° C.). **RT = room temperature. Both filtrations for thesame strain were performed from the same day harvest. Prior tofiltration through Sartoclean CA and Sartopore2 filters, VAF was exposedto 31 ± 3° C. for 0 (RT) or 60 minutes.

TABLE 24 Neuraminidase activity [μU/mL] of six influenza strains.Process step Stabilized Filtered Centrifuged Activity Influenza strainVAF VAF Stabilized VAF* Gain/Loss A/Beijing/262/95 RT** 56.5 47.5 54.5−7.0 A/Beijing/262/95 31 ± 3° C. 64.5 56.0 58.5 −1.5 A/NewCaledonia/20/99 RT** 46.0 38.5 40.0 −1.5 A/New Caledonia/20/99 31 ± 3°C. 46.0 43.0 40.0 3.0 A/Sydney/05/97 RT** 35.5 7.0 23.0 −16.0A/Sydney/05/97 31 ± 3° C. 35.5 14.5 23.0 −8.5 A/Panama/2007/99 RT** 55.515.0 34.5 −19.5 A/Panama/2007/99 31 ± 3° C. 60.5 42.5 39.0 3.5B/Victoria/504/2000 RT** 35.0 21.0 28.5 −7.5 B/Victoria/504/2000 31 ± 3°C. 39.0 25.5 31.5 −6.0 B/Yamanashi/166/98 RT** 29.0 26.0 28.5 −2.5B/Yamanashi/166/98 31 ± 3° C. 33.5 27.5 29.5 −2.0 *Stabilized VAF andCentrifuged Stabilized VAF (control) samples were taken from the poolbefore VAF was divided into individual experiments (RT and 31 ± 3° C.).**RT = room temperature. Both filtrations for the same strain wereperformed from the same day harvest. Prior to filtration throughSartoclean CA and Sartopore 2 filters, VAF was exposed to 31 ± 3° C. for0 (RT) or 60 minutes.

TABLE 25 Hemagglutinin activity [HA titer] of six influenza strains.Process step Stabilized Stabilized Warmed up Filtered CentrifugedInfluenza strain VAF* VAF VAF Stabilized VAF* A/Beijing/262/95 RT** 256— 1024 512 A/Beijing/262/95 31 ± 32° C. 256 1024  2048 512 A/NewCaledonia/20/99 RT** 512 — 512 512 A/New Caledonia/20/99 31 ± 3° C. 512512  512 512 A/Sydney/05/97 RT** 64 — 16 64 A/Sydney/05/97 31 ± 3° C. 6464 64 64 A/Panama/2007/99 RT** 256 — 64 256 A/Panama/2007/99 31 ± 3° C.256 512  512 256 B/Victoria/504/2000 RT** 64 — 128 128B/Victoria/504/2000 31 ± 3° C. 64 64 64 128 B/Yamanashi/166/98 RT** 128— 32 128 B/Yamanashi/166/98 31 ± 3° C. 128 64 64 128 *Stabilized VAF andCentrifuged Stabilized VAF (control) samples were taken from the poolbefore VAF was divided into individual experiments (RT and 31 ± 3° C.).**RT = room temperature. Both filtrations for the same strain wereperformed from the same day harvest. Prior to filtration throughSartoclean CA and Sartopore 2 filters, VAF was exposed to 31 ± 3° C. for0 (RT) or 60 minutes.

TABLE 26 Analysis by SEC - Peak Area Comparison Peak Area at 220 VirusImpurities Impurities Peak Group 1 Group 2 Sample Details Sample ID~(10.5 min) (18 to 21 min) (21 to 27 min) Neat (VH)  1X 1221 31785339528 10 times concentrated sample 10X 11192 126849 435652  1 × Washed5 times with 1X-SPG  1X-W 1005 2131 2510 10 × washed with 1X-SPG 5 times10X-W 10282 15858 2194 Permeate or filtrate Permeate 25 33837 360812Wash-1 W-1 6626 71260 Wash-2 W-2 2296 15773 Wash-3 W-3 1879 5765 Wash-4W-4 1046 3110 Wash-5 W-5 876 2769

TABLE 27 A/New Caledonia - CELISA Values Mean +/− SD Sample DetailsSample ID Replicate (N) (CELISA) Neat (VH)  1X 4  9.1 +/− 0.02 10 timesconcentrated sample 10X 4 10.0 +/− 0.05  1 × Washed 5 times with 1X-SPG 1X-W 4  8.9 +/− 0.03 10 × washed with 1X-SPG 5 times 10X-W 4  9.9 +/−0.04 Permeate or filtrate Permeate 4 <LOQ 10X diluted back to 1X with1X-SPG 10X to 1X 4  9.0 +/− 0.08 10X-W diluted back to 1X-W with 1X-SPG10X-W to 1X-w 4  8.9 +/− 0.02

TABLE 28 Composition of Representative Formulations Formulation NumberComposition 1 10% Allantoic fluid in 100 millimolar phosphate buffer, 7%Sucrose, no added excipients 2 60% Allantoic fluid in 100 millimolarphosphate buffer, 7% Sucrose, no added excipients 3 10% Allantoic fluidin 100 millimiolar phosphate buffer, 7% sucrose [2] with 1% gelatinhydrolysate and 1% arginine 4 60% Allantoic fluid in 100 millimolarphosphate buffer, 7% sucrose [3] with 1% gelatin hydrolysate and 1%arginine 5 60% Allantoic fluid in 100 millimolar phosphate buffer, 10%sucrose, 2% arginine, 2% gelatin hydrolysate 6 60% Allantoic fluid in100 millimolar phosphate buffer, 10% sucrose, 2% arginine 7 60%Allantoic fluid in 100 millimolar phosphate buffer, 10% sucrose, 2%arginine, 2% gelatin hydrolysate, 2.5 mM EDTA 8 60% Allantoic fluid in50 millimolar histidine buffer, 10% sucrose, 2% arginine, 2% gelatinhydrolysate 9 60% Allantoic fluid in 50 millimolar histidine buffer, 10%sucrose, 2% arginine, 2% gelatin hydrolysate, 2.5 mM EDTA

TABLE 29 Stability of Virus in Representative Formulations (loss oftiter in log₁₀/mL/month) Formulation A/New A/Panama/ B/Hong Kong/ NumberCaledonia20/99 2007/99 330/01 1 0.030 0.133 0.156 2 0.040 0.098 0.166 30.042 0.080 0.151 4 0.087 0.073 0.181 5 0.021 0.093 0.107 6 No lossobserved 0.090 0.097 7 0.046 0.037 0.113 8 0.068 0.072 0.061 9 0.0340.073 0.121

TABLE 30 INGRE- avs43 avs53 DIENTS Liq01 Liq02 Liq03 Liq04 Liq05 Liq06Liq07 Liq08 Liq09 Liq10 Liq11 Liq12 KPO4 100 mM 100 mM 100 mM 100 mM 100mM 100 mM 100 mM 100 mM 100 mM 100 mM PH 7.2 HEPES 100 mM Sucrose 15%15% 15% 15% 10% 15% 15% 15% 20% 20% 20% Gelatin  1%  1%  1%  2%  2%  1% 2%  2%  2% Arginine  2%  2%  2%  2%  2%  2%  2% Glycine  1% Methi-0.15%   onine PVP  1% Dextran  1% Pluronic 0.02%   0.02%   CAIV 10% 10%10% 10% 10% 10% 10% 10% 10% 10% 10% 10% Mono- valent (added) NAF 50% 50%50% 50% 50% 50% 50% 50% 50% 50% 50% 50% (added) Purified q.s. q.s. q.s.q.s. q.s. q.s. q.s. q.s. q.s. q.s. q.s. q.s. Water

TABLE 31 B/Hongkong (in SPG) Target: 6.9 4° C. (Monthly) Formulation 0 01 1 2 2 3 3 4 4 5 5 6 6 Formulation Liq 01 7.3 7.3 6.6 6.6 6.1 6.2 6.56.5 6.5 6.6 6.7 6.7 6.5 6.4 15Suc/1Gel/2Arg Liq 01 (Repeat) 6.9 6.8 6.76.6 6.6 6.5 6.6 6.5 6.4 6.4 6.4 15Suc/1Gel/2Arg Liq 02 7.3 7.3 6.6 6.46.3 6.4 6.3 6.3 6.4 6.4 6.6 6.7 6.1 6.3 15Suc/1Gel/1Glyc Liq 02 (Repeat)6.7 6.6 6.3 6.6 6.5 6.6 6.4 6.3 6.4 6.3 6.2 6.2 15Suc/1Gel/1Glyc Liq 037.3 7.1 6.5 6.5 6.2 6.1 6.4 6.3 6.4 6.2 6.7 6.8 6.3 15Suc/1Gel Liq 03(Repeat) 6.8 6.9 6.7 6.7 6.5 6.6 6.4 6.4 6.3 6.2 6.3 6.2 15Suc/1Gel Liq04 7.2 7.2 6.3 6.5 6.1 6.1 6.2 6.4 6.4 6.4 6.7 6.8 6.5 6.315Suc/2Gel/2Arg Liq 04 (Repeat) 6.8 6.8 6.7 6.7 6.5 6.5 6.4 6.4 6.4 6.46.3 6.2 15Suc/2Gel/2Arg Liq 05 7.1 7.1 6.5 6.5 6.1 6.1 6.4 6.2 6.4 6.56.7 6.7 6.4 6.2 10Suc/2Gel/2Arg Liq 05 (Repeat) 7.0 7.1 6.8 6.6 6.6 6.66.7 6.9 6.7 6.8 6.9 6.9 10Suc/2Gel/2Arg Liq 06 7.1 7.0 5.8 5.7 5.4 5.35.1 5.3 4.6 4.1 4.4 3.6 3.7 5.0 15Suc/1Gel/2Arg, HEPES Liq 07 7.0 7.16.3 6.3 5.9 5.9 6.3 6.3 6.2 6.2 6.5 6.6 6.4 6.4 15Suc/1PVP/2Arg Liq 07(Repeat) 6.7 6.6 6.4 6.5 6.3 6.4 6.4 6.3 6.3 6.2 6.1 6.2 15Suc/1PVP/2ArgLiq 08 7.0 6.9 6.4 6.4 6.0 6.0 6.3 6.4 6.4 6.1 7.1 6.7 6.4 6.315Suc/1Dextran/2Arg Liq 08 (Repeat) 6.7 6.8 6.6 6.7 5.8 6.7 6.4 6.6 6.46.4 6.2 6.2 15Suc/1Dextran/2Arg Liq 09 6.9 7.0 6.5 6.4 6.0 6.0 6.3 6.36.0 6.2 6.7 6.6 6.3 6.4 20Suc/2Gel/2Arg Liq 09 (Repeat) 6.9 6.9 6.7 6.86.6 6.6 6.6 6.6 6.3 6.4 6.4 6.3 20Suc/2Gel/2Arg Liq 10 6.9 7.0 6   6.35.7 6.0 6.2 6.3 5.9 6.2 6.7 6.5 6.2 6.3 20Suc/2 Ge//0.15 Meth/0.02PlurLiq 10 (Repeat) 6.9 6.9 6.5 6.5 6.5 6.3 6.3 6   6.2 6.2 6.2 6   20Suc/2Ge//0.15 Meth/0.02Plur Liq 11 7.0 7.0 6.3 6.3 6.0 5.9 6.2 6.4 6.0 6.06.4 6.6 6.1 6.1 20Suc/2Ge//0.02Plur Liq 12 6.9 7.0 6.3 6.5 6.4 6.4 6.16.1 5.8 5.3 5.1 5.3 5.6 5.3 NAF only (60%)

TABLE 32 B/Harbin (in SPG) Target: 7.0 4° C. (Monthly) Formulation 0 0 11 2 2 3 3 4 4 5 5 6 6 Formulation Liq 01 7.1 7.2 6.6 6.7 6.2 6.4 6.6 6.66.8 6.8 6.7 6.8 6.2 6.1 15Suc/1Gel/2Arg Liq 01 (Repeat) 6.9 6.9 6.7 6.86.7 6.6 6.6 6.6 6.6 6.4 6.4 6.5 15Suc/1Gel/2Arg Liq 02 7.1 7.1 6.7 6.66.2 6.0 6.5 6.5 6.5 6.7 6.7 6.7 6.0 6.0 15Suc/1Gel/1Glyc Liq 02 (Repeat)6.9 7   6.8 6.7 6.5 6.6 6.5 6.5 6.3 6.4 6.3 6.2 15Suc/1Gel/1Glyc Liq 037.1 7.0 6.5 6.7 6.1 6.0 6.5 6.5 6.7 6.5 6.7 7.1 6.2 6.3 15Suc/1Gel Liq03 (Repeat) 6.9 6.9 6.7 6.7 6.5 6.5 6.6 6.5 6.5 6.4 6.4 6.4 15Suc/1GelLiq 04 7.0 7.0 6.5 6.8 6.1 6.2 6.5 6.6 7.0 6.9 7.3 7.1 6.4 6.515Suc/2Gel/2Arg Liq 04 (repeat) 6.8 6.8 6.7 6.7 6.7 6.6 6.5 6.5 6.4 6.56.4 6.5 15Suc/2Gel/2Arg Liq 05 7.1 7.2 6.8 6.7 6.2 6.1 6.6 6.7 6.9 6.76.9 6.9 6.4 6.3 10Suc/2Gel/2Arg Liq 05 (Repeat) 7.0 7.0 6.6 6.5 6.7 6.86.8 6.8 6.7 6.8 6.7 6.6 10Suc/2Gel/2Arg Liq 06 7.1 7.1 6.4 6.3 5.4 5.45.4 5.3 5.4 5.2 4.3 4.3 3.8 ud 15Suc/1Gel/2Arg, HEPES Liq 07 7.0 7.1 6.76.8 6   6.2 6.4 6.4 6.5 6.6 6.4 6.7 6.4 6.5 15Suc/1PVP/2Arg Liq 07(Repeat) 6.9 6.8 6.6 6.5 6.5 6.6 6.4 6.4 6.3 6.2 6.4 6.4 15Suc/1PVP/2ArgLiq 08 7.1 6.9 6.6 6.9 5.9 6.1 6.4 6.4 6.6 6.9 6.7 6.7 6.2 6.415Suc/1Dextran/2Arg Liq 08 (Repeat) 6.8 6.9 6.7 6.6 6.6 6.6 6.4 6.4 6.26.4 6.4 6.3 15Suc/1Dextran/2Arg Liq 09 6.9 7.0 6.8 6.9 6.1 6.2 6.5 6.56.8 6.8 6.6 6.9 6.4 6.4 20Suc/2Gel/2Arg Liq 09 (Repeat) 6.7 6.7 6.6 6.56.4 6.4 6.3 6.2 6.4 6.2 6.5 6.6 20Suc/2Gel/2Arg Liq 10 7.0 7.0 ud ud 6.16.2 6.4 6.4 6.7 6.8 6.5 6.6 6.6 6.4 20Suc/2 Ge//0.15 Meth/0.02Plur Liq10 (Repeat) 6.8 7.0 6.7 6.7 6.6 6.5 6.5 6.3 6.4 6.4 6.3 6.3 20Suc/2Ge//0.15 Meth/0.02Plur Liq 11 6.9 6.9 ud ud 6   6.1 6.4 6.5 6.6 6.7 6.66.3 6.5 6.4 20Suc/2Ge//0.02Plur Liq 12 6.9 6.9 6.5 6.5 6.3 6.1 6.0 6.16.1 6.1 5.8 5.7 5.9 5.9 NAF only (60%)

TABLE 33 A/New Caledonia (in SPG) Target: 6.8 4° C. (Monthly)Formulation 0 0 1 1 2 2 3 3 4 4 5 5 6 6 Formulation Liq 01 7.1 7.0 7.07.0 6.6 6.8 6.5 6.6 6.7 6.8 6.9 6.8 6.5 6.6 15Suc/1Gel/2Arg Liq 01(Repeat) 7.0 7.0 6.9 6.7 6.7 6.6 6.5 6.5 6.6 6.7 6.6 6.6 15Suc/1Gel/2ArgLiq 02 7.0 7.0 6.9 7.0 6.7 6.6 6.6 6.4 6.5 6.6 6.9 6.8 6.3 6.415Suc/1Gel/1Glyc Liq 02 (Repeat) 7.1 7.1 6.9 6.9 6.9 6.7 6.8 6.7 6.7 6.86.7 6.4 15Suc/1Gel/1Glyc Liq 03 7.0 7.2 6.8 6.9 6.6 6.4 6.5 6.5 6.5 6.56.9 6.9 6.4 6.4 15Suc/1Gel Liq 03 (Repeat) 6.8 6.9 6.7 6.7 6.6 6.6 6.56.6 6.6 6.6 6.6 6.5 15Suc/1Gel Liq 04 7.2 7.2 6.8 7.0 6.7 6.4 6.5 6.56.5 6.7 7.0 7.1 6.5 6.5 15Suc/2Gel/2Arg Liq 04 (Repeat) 6.9 7.0 6.8 6.96.8 6.9 6.8 6.8 6.7 6.6 6.5 6.4 15Suc/2Gel/2Arg Liq 05 7.2 7.1 7.0 7.06.7 6.6 6.5 6.6 6.6 6.7 7.0 7.1 6.7 6.6 10Suc/2Gel/2Arg Liq 05 (repeat)6.9 6.9 6.9 6.8 6.8 6.8 6.8 6.6 6.7 6.6 6.6 10Suc/2Gel/2Arg Liq 06 6.96.9 6.4 6.3 5.5 5.5 5.4 5.0 UD UD 4.7 4.7 3.8 4.1 15Suc/1Gel/2Arg, HEPESLiq 07 7.1 7.1 6.5 6.6 6   6.1 6.3 6.3 6.3 6.3 6.8 6.6 6.4 6.215Suc/1PVP/2Arg Liq 07 (Repeat) 6.9 7.0 6.4 6.5 6.3 6.4 6.6 6.5 6.4 6.36.5 6.2 15Suc/1PVP/2Arg Liq 08 7.1 6.9 6.8 6.9 6.3 6.4 6.4 6.4 6.5 6.56.8 7.3 6.4 6.4 15Suc/1Dextran/2Arg Liq 08 (Repeat) 6.7 6.7 6.6 6.5 6.56.5 6.5 6.6 6.3 6.5 6.5 6.3 15Suc/1Dextran/2Arg Liq 09 6.8 6.9 7.0 6.86.5 5.7 6.5 6.5 6.4 6.5 7.1 7.2 6.4 6.3 20Suc/2Gel/2Arg Liq 09 (Repeat)6.8 6.9 6.8 6.7 6.6 6.6 6.7 6.6 6.5 6.6 6.6 6.3 20Suc/2Gel/2Arg Liq 106.7 6.9 6.8 6.8 6.6 6.5 6.4 6.3 6.2 6.2 6.9 7.0 6.3 6.3 20Suc/2 Ge//0.15Meth/0.02Plur Liq 10 (Repeat) 6.6 6.6 6.5 6.5 6.4 6.4 6.3 6.3 6.5 6.36.2 6.2 20Suc/2 Ge//0.15 Meth/0.02Plur Liq 11 6.8 6.7 6.8 6.7 6.7 6.66.3 6.4 6.3 6.2 6.7 6.8 6.4 6.3 20Suc/2Ge//0.02Plur Liq 12 6.9 7.0 5.75.6 5.5 5.3 5.1 5.3 4.7 4.6 4.5 4.5 4.4 4.5 NAF only (60%)

TABLE 34 A/Panama (in SPG) Target: 7.4 4° C. (Monthly) Formulation 0 0 11 2 2 3 3 4 4 5 5 6 6 Formulation Liq 01 7.6 7.6 7.4 7.3 6.9 7.1 6.6 6.66.8 7.1 7.4 7.4 6.6 6.7 15Suc/1Gel/2Arg Liq 01 (Repeat) 6.8 6.8 6.8 6.66.6 6.7 6.7 6.8 6.6 6.6 6.7 6.7 15Suc/1Gel/2Arg Liq 02 7.6 7.6 7.3 7.36.9 6.4 6.6 6.6 6.9 6.8 7.3 7.4 6.6 6.6 15Suc/1Gel/1Glyc Liq 02 (Repeat)7.3 7.0 7.3 7.1 7.1 7.4 7.3 7.2 7.2 6.7 6.7 15Suc/1Gel/1Glyc Liq 03 7.27.8 7.2 7.3 6.8 6.7 6.6 6.7 6.6 7.1 7.3 7.4 6.6 6.8 15Suc/1Gel Liq 03(Repeat) 6.8 6.8 6.9 6.8 6.7 6.8 6.8 6.8 6.7 6.8 6.8 6.6 15Suc/1Gel Liq04 7.8 7.8 7.4 7.4 7.1 7.1 6.5 6.6 6.9 6.9 7.5 7.4 6.6 6.715Suc/2Gel/2Arg Liq 04 (Repeat) 7.0 7.1 6.9 6.9 6.8 6.9 6.6 6.8 6.7 6.66.7 6.8 15Suc/2Gel/2Arg Liq 05 7.8 7.8 7.5 7.4 6.8 6.8 6.7 6.7 7.0 7.07.4 7.3 6.7 6.7 10Suc/2Gel/2Arg Liq 05 (repeat) 7.0 7.0 6.6 6.5 6.7 6.86.8 6.8 6.7 6.8 6.7 6.6 10Suc/2Gel/2Arg Liq 06 7.7 7.6 6.9 7.0 6.3 6.46.2 5.9 5.8 5.8 6.0 6.0 5.0 4.8 15Suc/1Gel/2Arg, HEPES Liq 07 7.2 7.17.4 7.0 7.0 6.6 6.7 6.5 7.0 7.1 7.2 7.3 6.6 6.3 15Suc/1PVP/2Arg Liq 07(Repeat) 7.2 7.2 7.2 6.9 7.2 6.9 6.8 6.6 6.6 6.7 6.8 6.9 15Suc/1PVP/2ArgLiq 08 7.1 7.2 7.1 7.2 6.6 6.8 6.7 6.8 6.9 6.9 7.2 7.2 6.5 6.615Suc/1Dextran/2Arg Liq 08 (Repeat) 7.2 6.8 7.0 6.8 6.9 6.9 6.9 6.5 6.86.9 6.9 6.7 15Suc/1Dextran/2Arg Liq 09 7.4 7.3 6.9 6.9 6.7 7.0 6.3 6.86.7 7.0 7.3 7.3 6.5 6.5 20Suc/2Gel/2Arg Liq 09 (Repeat) 7.2 7.2 7.0 6.76.8 7.0 6.9 6.9 6.7 7.0 6.8 6.7 20Suc/2Gel/2Arg Liq 10 7.2 7.1 7.1 7.16.8 6.6 6.6 6.4 6.7 6.7 7.3 7.2 6.5 6.6 20Suc/2 Ge//0.15 Meth/0.02PlurLiq 10 (Repeat) 7.0 6.7 6.7 6.7 6.7 6.7 6.6 6.6 6.6 6.6 6.6 6.5 20Suc/2Ge//0.15 Meth/0.02Plur Liq 11 7.1 6.9 7.1 7.2 6.5 6.6 6.7 6.5 6.6 6.97.2 7.3 6.6 6.4 20Suc/2Ge//0.02Plur Liq 12 7.2 7.3 6   6.4 6.3 5.9 4.95.0 5.1 4.5 4.6 4.6 3.8 4.1 NAF only (60%)

TABLE 35 Control Liq15 Ingredients Liq13a Liq14 Liq15 (degassed) Liq16Liq17 Liq18 Liq19 Liq20 KPO4 buffer, pH 7.2 1.1 mM 100 mM 100 mM 100 mM100 mM 100 mM 100 mM 100 mM (1.1 mM from virus included) Citrate buffer,pH 7.2 100 mM Sucrose (0.7% from  7% 10% 10% 10% 10% 10% 10% 10% 10%virus included) Gelatin  2%  2%  2%  2%  2%  2%  2%  2% Arginine  2%  2% 2%  2%  2%  2%  2%  2% Aprotinin (PI) 0.02%   Leupeptin 0.02%  hemisulfate (PI) Lysozyme Inhibitor 0.1%  Protease Inhibitor (0.6%Cocktail DMSO) 0.5%* PMSF  1 mM Cytidine 2′ monophosphate NAF (fromvirus) 10% 10% 10% 10% 10% 10% 10% 10% 10% NAF (added) 50% 50% 50% 50%50% 50% 50% 50% 50% 1N KOH or iN HCl pH 7.2 to pH 7.0 to pH 7.2 to pH7.2 to pH 7.2 to pH 7.2 to pH 7.2 to pH 7.2 to pH 7.2 to pH 7.2 PurifiedWater None q.s. q.s. q.s. q.s. q.s. q.s. q.s. q.s. added IngredientsLiq21 Liq22 Liq23 Liq31 Liq24 Liq25 Liq26 Liq27 KPO4 buffer, pH 7.2 100mM 100 mM 100 mM 100 mM 100 mM 100 mM 100 mM 100 mM (1.1 mM from virusincluded) Sucrose (0.7% from 10% 10% 10% 10% 10% 10% 9.3% virusincluded) Gelatin 10%  2%  2%  2%  2%  2%  2%  2% Arginine  2%  2%  2% 2%  2%  2%  2%  2% L-Ascorbic Acid  2% 0.05%   Ascorbic Acid 6 0.005%  0.001%   Palmitate Arbutin 0.05%   Propyll Gallate 0.05%   EDTA  10 mMRNAse Inhibitor, (0.05% Glyc) SuperAse In 2.0 U/μL NAF (from virus)  5mM 10% 10% 10% 10% 10% 10% 10% NAF (added) 10% 50% 50% 50% 50% 50% 50%50% 1N KOH or 1N HCl 50% titrate titrate titrate titrate titrate titratetitrate to pH 7.2 to pH 7.2 to pH 7.2 to pH 7.2 to pH 7.2 to pH 7.2 topH 7.2 to pH 7.2 Purified Water to pH 7.2 q.s. q.s. q.s. q.s. q.s. q.s.q.s. q.s.

TABLE 36 SP stabilized B/Hongkong Target: 6.9 15° C. (weekly) 0 0 2 2 44 6 6 8 8 10 10 12 12 14 14 Liq 13a 6.6 6.7 6.3 6.3 5.8 5.8 5.7 5.6 5.55.0 5.1 5.2 5.5 5.1 4.4 4.7 no excipient (60% NAF), equil. at 15° C. Liq13b 6.6 6.6 6.4 6.4 6.0 5.8 5.6 5.5 5.0 5.3 4.8 4.9 ud 4.6 ud 5.2 noexcipient (60% NAF), equil. at 33° C. Liq 13c 6.5 6.7 6.1 6.3 5.7 5.85.5 5.6 5.3 5.2 5.1 4.7 4.7 4.5 4.7 4.5 no excipient (60% NAF), equil.at 45° C. Liq 14 6.7 6.8 6.4 6.7 6.0 6.0 5.7 5.6 5.3 5.3 5.3 5.3 5.0 4.94.8 4.8 Citr., 10Suc/2Gel/2Arg, equil. at 15° C. Liq 15a 6.9 6.7 6.6 6.56.2 6.2 6.0 5.9 5.6 5.8 5.5 5.5 5.4 5.1 5.0 5.4 10Suc/2Gel/2Arg, equil.at 15° C. Liq 15b 6.7 6.9 6.6 6.5 6.1 6.1 6.0 6.0 5.4 5.3 4.8 5.7 5.15.2 5.0 4.7 10Suc/2Gel/2Arg, equil. at 33° C. Liq 15c 6.7 6.8 6.4 6.45.9 6.0 5.7 5.8 4.8 5.3 5.2 5.2 ud 5.0 4.5 4.7 10Suc/2Gel/2Arg, equil.at 45° C. Liq 15d 7.0 7.0 6.2 6.3 5.7 5.9 5.8 5.8 disc. 10Suc/2Gel/2Arg(Degassed) Liq 16 6.8 6.9 6.2 6.2 5.8 5.8 5.8 5.5 5.6 5.6 5.6 5.2 disc.10Suc/2Gel/2Arg/0.02Aprotinin(PI) Liq 17 6.8 6.9 6.1 6.1 5.8 5.8 5.6 5.95.6 5.7 5.6 5.5 disc. 10Suc/2Gel/2Arg/0.02Leup.Hemisulfate(PI) Lip 187.0 7.1 6.3 6.3 6.2 6.0 5.9 5.9 disc. 10Suc/2Gel/2Arg/0.1Lysozyme Inhib.Liq 19 6.6 6.6 6.2 6.2 5.9 5.6 5.7 5.6 5.7 5.5 5.3 5.2 disc.10Suc/2Gel/2Arg/0.5Prot.Inhib.Cocktail Liq 20 ud 4.8 ud ud ud ud dsic.10Suc/2Gel/2Arg/1 mM PMSF Liq 21 6.7 6.5 6.3 6.3 5.9 5.9 5.9 5.8 6.3 6.35.8 5.4 disc. 10Suc/2Gel/2Arg/1 mM Cytid.2′Monophos. Liq 22 6.8 6.7 5.85.6 5.6 5.7 ud ud ud ud disc. 10Suc/2Gel/2Arg/0.05L-Ascorbic Acid Liq22a 6.5 6.5 5.2 5.9 disc. 10Suc/2Gel/2Arg/0.05L-Ascorbic Acid (deg) Liq23 4.5 4.9 ud ud ud ud disc 10Suc/2Gel/2Arg/0.005AscorbAcid6PalmitateLiq 24 6.8 6.7 6.4 6.5 6.3 5.9 5.8 5.7 5.3 5.5 5.8 5.3 5.1 5.1 4.8 4.810Suc/2Gel/2Arg/0.05Arbutin Liq 24 (deg) 6.7 6.6 6.2 6.3 6.0 5.0 disc.10Suc/2Gel/2Arg/0.05Arbutin Liq 25 6.6 6.7 5.1 6.2 5.1 ud ud ud ud uddisc. 10Suc/2Gel/2Arg/0.05PropyllGallate Liq 25 (deg) 6.4 6.4 4.8 4.7disc. 10Suc/2Gel/2Arg/0.05PropyllGallate Liq 26 6.8 6.8 6.6 6.4 6.3 6.35.8 6.0 5.4 5.5 5.4 5.6 5.3 5.4 5.3 5.3 10Suc/2Gel/2Arg/10 mM EDTA Liq26a 6.8 6.7 6.5 6.5 6.5 6.3 10Suc/2Gel/2Arg/10 mM EDTA (deg) Liq 27 6.66.7 ud ud 10Suc/2Gel/2Arg/RNAse Inhibt. 2 U/μL Liq 31 ud 5.9 ud ud ud uddisc 10Suc/2Gel/2Arg/0.001 AscorbAcid6Palmitate

TABLE 37 SP stabilized A/Panama Target: 7.5 15° C. (weekly) 0 0 2 2 4 46 6 8 8 10 10 12 12 14 14 16 16 Liq 13a 7.0 6.9 6.4 6.4 5.3 5.5 5.4 5.35.3 4.7 5.1 4.8 4.5 4.6 4.2 3.8 no excipient (60% NAF), equil. at 15° C.Liq 13b 6.9 6.9 6.4 6.3 5.4 5.6 5.4 5.6 5.2 4.8 4.9 5.0 4.8 4.6 4.3 4.6no excipient (60% NAF), equil. at 33° C. Liq 13c 6.8 6.7 6.3 6.3 5.4 5.65.3 5.0 5.1 4.7 4.5 4.7 4.7 4.9 4.5 4.2 no excipient (60% NAF), equil.at 45° C. Liq 14 7.3 7.3 7.2 7.2 6.8 6.8 6.7 6.8 6.5 6.5 6.7 6.6 6.4 6.56.1 6.2 6.5 6.5 Citr., 10Suc/2Gel/2Arg, equil. at 15° C. Liq 15a 7.2 7.26.9 7   6.6 6.7 6.6 6.4 6.2 6.0 6.0 6.0 5.7 6.1 5.7 5.7 6.1 5.810Suc/2Gel/2Arg, equil. at 15° C. Liq 15b 7.3 7.1 7.0 6.9 6.3 6.5 6.76.6 6.0 5.8 6.1 6.0 6.1 5.8 5.9 5.8 6.0 6.0 10Suc/2Gel/2Arg, equil. at33° C. Liq 15c 7.1 6.9 6.8 6.8 6.3 6.0 6.3 6.5 5.8 5.8 6.1 6.1 5.8 5.85.8 5.8 5.9 5.9 10Suc/2Gel/2Arg, equil. at 45° C. Liq 15d 6.8 6.7 6.76.5 6.5 6.4 6.4 6.4 6.4 6.4 10Suc/2Gel/2Arg (Degassed) Liq 16 only forB/ 10Suc/2Gel/2Arg/ Hongkong 0.02Aprotinin(PI) Liq 17 only for B/10Suc/2Gel/2Arg/ Hongkong 0.02Leup.Hemisulfate(PI) Liq 18 6.9 6.9 6.76.5 6.2 6.5 6.2 6.4 6.5 6.4 10Suc/2Gel/2Arg/ 0.1Lysozyme Inhib. Liq 196.5 6.6 6.6 6.5 5.7 6.0 5.9 ud 5.4 5.4 5.5 5.8 5.8 5.8 10Suc/2Gel/2Arg/0.5Prot.Inhib.Cocktail Liq 20 6.8 6.9 6.0 5.7 ud ud ud ud disc.10Suc/2Gel/2Arg/1 mM PMSF Liq 21 6.9 6.7 6.2 6.3 6.5 6.6 6.9 7.1 5.8 5.96.1 6.0 5.9 5.7 6   6.1 5.8 5.6 10Suc/2Gel/2Arg/ 1 mM Cytid.2′Monophos.Liq 22 7.1 7.2 6.0 6.0 5.9 5.8 5.1 ud 5.7 ud ud 5.4 ud ud disc.10Suc/2Gel/2Arg/ 0.05L-Ascorbic Acid Liq 22a 6.7 6.7 6.2 5.9 6.0 5.910Suc/2Gel/2Arg/ (deg) 0.05L-Ascorbic Acid Liq 23 5.6 5.5 ud ud ud disc.10Suc/2Gel/2Arg/ 0.005AscorbAcid6Palmitate Liq 24 7.0 6.9 6.7 6.8 6.66.6 6.4 6.5 6.2 6.2 6   6.3 6.1 5.9 5.7 5.9 5.9 6.110Suc/2Gel/2Arg/0.05Arbutin Liq 24a 6.6 6.6 6.5 6.610Suc/2Gel/2Arg/0.05Arbutin (deg) Liq 25 6.9 6.5 5.1 5.5 ud ud ud ud udud disc 10Suc/2Gel/2Arg/ 0.05PropyllGallate Liq 25a ud ud ud ud disc10Suc/2Gel/2Arg/ (deg) 0.05PropyllGallate Liq 26 7.4 7.3 7.2 7.1ud(repd) ud(repd) 6.9 6.9 6.7 6.7 6.7 6.9 6.5 6.4 6.4 6.410Suc/2Gel/2Arg/10 mM EDTA Liq 26a 7.1 7.2 7   7.1 7.1 6.910Suc/2Gel/2Arg/10 mM (deg) EDTA Liq 27 only for B/10Suc/2Gel/2Arg/RNAse Hongkong Inhibt. 2 U/μL Liq 31 ud ud ud ud 3.5 3.8disc. 10Suc/2Gel/2Arg/ 0.001AscorbAcid6Palmitate

TABLE 38 SP stabilized B/Hongkong Target: 6.9 4° C. (Monthly) 0 0 1 1 22 3 3 4 4 Liq 13a 6.6 6.7 6.4 6.5 6.2 6.2 6.4 6.3 6.1 6.2 no excipient(60% NAF), equil. at 15° C. Liq 13b 6.6 6.6 6.5 6.4 6.3 6.2 6.3 6.4 6.16.2 no excipient (60% NAF), equil. at 33° C. Liq 13c 6.5 6.7 6.4 6.4 6.16.1 6.3 6.4 6   5.9 no excipient (60% NAF), equil. at 45° C. Liq 14 6.06.8 6.4 6.5 6.0 6.4 6.3 6.4 6.1 6.1 Citr., 10Suc/2Gel/2Arg, equil. at15° C. Liq 15a 6.9 6.7 6.7 6.8 6.4 6.3 6.7 6.7 6.5 6.5 10Suc/2Gel/2Arg,equil. at 15° C. Liq 15b 6.7 6.9 6.7 6.6 6.0 6.2 6.5 6.5 6.4 6.410Suc/2Gel/2Arg, equil. at 33° C. Liq 15c 6.7 6.8 6.6 6.6 6.1 6.2 6.56.4 6.4 6.4 10Suc/2Gel/2Arg, equil. at 45° C. Liq 15d 7.0 7.0 6.8 6.86.5 6.3 10Suc/2Gel/2Arg (Degassed) Liq 16 6.8 6.9 6.4 6.7 6.6 6.5 6.16.3 10Suc/2Gel/2Arg/0.02Aprotinin(PI) Liq 17 6.8 6.9 6.7 6.6 6.5 6.5 6.16.3 10Suc/2Gel/2Arg/0.02Leup.Hemisulfate(PI) Liq 18 7.0 7.1 6.9 6.8 6.56.5 10Suc/2Gel/2Arg/0.1Lysozyme Inhib. Liq 19 6.6 6.6 6.5 6.4 6.5 6.510Suc/2Gel/2Arg/0.5Prot.Inhib.Cocktail Liq 20 ud 4.8 ud ud disc.10Suc/2Gel/2Arg/1 mM PMSF Liq 21 6.7 6.5 6.6 6.6 7.0 7.0 6.6 6.610Suc/2Gel/2Arg/1 mM Cytid.2′Monophos. Liq 22 6.8 6.7 5.9 UD UD UD 5.25.5 10Suc/2Gel/2Arg/0.05L-Ascorbic Acid Liq 22a (deg) 6.5 6.5 6   6.210Suc/2Gel/2Arg/0.05L-Ascorbic Acid Liq 23 4.5 4.9 ud ud disc.10Suc/2Gel/2Arg/0.005AscorbAcid6Palmitate Liq 24 6.8 6.7 6.7 6.6 ud(rep) ud (rep) 6.4 6.3 6.3 6.2 10Suc/2Gel/2Arg/0.05Arbutin Liq 24 (deg)6.7 6.6 7.0 7.0 10Suc/2Gel/2Arg/0.05Arbutin Liq 25 6.6 6.7 6.2 6.3 5.25.2 5.4 5.3 10Suc/2Gel/2Arg/0.05PropyllGallate Liq 25 (deg) 6.4 6.4 5.85.9 10Suc/2Gel/2Arg/0.05PropyllGallate Liq 26 6.8 6.8 6.6 6.7 6.6 6.56.6 6.6 6.3 6.3 10Suc/2Gel/2Arg/10 mM EDTA Liq 26a (deg) 6.8 6.7 6.9 6.910Suc/2Gel/2Arg/10 mM EDTA Liq 27 6.6 6.7 6.6 6.4 6.6 6.5 6.3 6.310Suc/2Gel/2Arg/RNAse Inhibt. 2 U/μL Liq 31 ud 5.9 5.1 4.9 disc.10Suc/2Gel/2Arg/0.001AscorbAcid6Palmitate

TABLE 39 SP stabilized A/Panama Target: 7.5 4° C. (Monthly) 0 0 1 1 2 23 3 4 4 Liq 13a 7.0 6.9 6.2 6.2 6.0 6.1 5.8 5.9 5.8 5.6 no excipient(60% NAF),equil. at 15° C. Liq 13b 6.9 6.9 5.9 6.0 6.0 6.0 5.9 5.7 5.96.0 no excipient (60% NAF), equil. at 33° C. Liq 13c 6.8 6.7 6   6.1 5.86.1 5.9 5.7 5.8 5.6 no excipient (60% NAF), equil. at 45° C. Liq 14 7.37.3 7.2 7.2 7.3 7.2 7.1 7.1 6.8 7   Citr., 10Suc/2Gel/2Arg, equil. at15° C. Liq 15a 7.2 7.2 6.7 6.6 6.6 6.6 6.5 7.0 6.4 6.6 10Suc/2Gel/2Arg,equil. at 15° C. big 15b 7.3 7.1 6.7 6.7 6.7 6.6 6.9 6.8 6.4 6.610Suc/2Gel/2Arg, equil. at 33° C. Liq 15c 7.1 6.9 6.5 6.4 6.7 6.6 6.66.6 6.4 6.4 10Suc/2Gel/2Arg, equil. at 45° C. Liq 15d 6.8 6.7 6.9 6.86.4 6.4 10Suc/2Gel/2Arg (Degassed) Liq 16 only for B/10Suc/2Gel/2Arg/0.02Aprotinin(PI) Hongkong Liq 17 only for B/10Suc/2Gel/2Arg/0.02Leup.Hemisulfate(PI) Hongkong Liq 18 6.9 6.9 6.9 6.86.4 6.3 10Suc/2Gel/2Arg/0.1Lysozyme Inhib. Liq 19 6.5 6.6 6.2 6.4 6.36.6 6.3 6.3 10Suc/2Gel/2Arg/0.5Prot.Inhib.Cocktail liq 20 6.8 6.9 6.97.0 7.1 7.2 6.9 6.7 10Suc/2Gel/2Arg/1 mM PMSF Liq 21 6.9 6.7 6.7 6.8 6.76.8 7.0 7.1 10Suc/2Gel/2Arg/1 mM Cytid.2′Monophos. Liq 22 7.1 7.2 6.26.0 6.3 5.8 6.0 5.9 6.1 6.1 10Suc/2Gel/2Arg/0.05L-Ascorbic Acid Liq 22a(deg) 6.7 6.7 6.7 6.6 10Suc/2Gel/2Arg/0.05L-Ascorbic Acid Liq 23 5.6 5.5ud ud disc. 10Suc/2Gel/2Arg/0.005AscorbAcid6Palmitate Liq 24 7.0 6.9 6.56.7 6.4 6.3 6.8 6.7 6.4 6.5 10Suc/2Gel/2Arg/0.05Arbutin Liq 24a (deg)6.7 6.6 6.2 6.2 10Suc/2Gel/2Arg/0.05Arbutin Liq 25 6.9 6.5 5.1 7.2 5.1ud disc. 10Suc/2Gel/2Arg/0.05PropyllGallate Liq 25a (deg) 6   6.3 rep6.4 10Suc/2Gel/2Arg/0.05PropyllGallate Liq 26 7.4 7.3 7.3 7.3 7.3 7.37.4 7.3 7.0 7.1 10Suc/2Gel/2Arg/10 mM EDTA Liq 26a (deg) 7.1 7.2 7.4 7.57.1 7.0 10Suc/2Gel/2Arg/10 mM EDTA Liq 27 only for B/10Suc/2Gel/2Arg/RNAse Inhibt. 2 U/μL Hongkong Liq 31 ud ud ud ud disc.10Suc/2Gel/2Arg/0.001AscorbAcid6Palmitate

TABLE 40 Formulation No 20 mM 50 mM 100 mM 200 mM Ingredients CitrateCitrate Citrate Citrate Citrate Citrate buffer 0  20 mM  50 mM  100 mM 200 mM pH 7.2 KPO4 buffer 1.1 mM 1.1 mM 1.1 mM  1.1 mM  1.1 mM (fromvirus material) Sucrose (0.7% 10% 10% 10% 10% 10% from virus included)Gelatin  1%  1%  1%  1%  1% Arginine  2%  2%  2%  2%  2% NAF (from 10%10% 10% 10% 10% virus: B/Hongkong; A/Panama) NAF (added) 50% 50% 50% 50%50% 1N KOH or 1N titrate titrate titrate titrate titrate HCl to pH 7.2to pH to pH 7.2 to pH 7.2 to pH 7.2 to pH 7.2 7.2 Purified Water q.s.q.s. q.s. q.s. q.s.Results: Potency by FFA Assay

TABLE 41 A/Panama A/Panama B/Hongkong B/Hongkong Aliquot 1 Aliquot 2Aliquot 1 Aliquot 2 A/Panama, Starting material Ave. of 9 Ave. of 6plates plates 8.1 ± 0.2 7.9 ± 0.1 (8.5) (7.9) Pre-diluted StartingMaterial (1:10) 8.0 ± 0.1 none n/a n/a (8.0) 0% Citrate 6.7 ± 0.1 6.9 ±0.0 6.7 ± 0.0 6.8 ± 0.1 (7.0) (7.0) (6.9) (6.9)  20 mM Citrate 6.7 ± 0.16.7 ± 0.2 6.9 ± 0.0 6.8 ± 0.1 (7.0) (7.0) (6.9) (6.9)  50 mM Citrate 6.7± 0.1 6.7 ± 0.1 6.9 ± 0.0 6.8 ± 0.1 (7.0) (7.0) (6.9) (6.9) 100 mMCitrate 6.8 ± 0.0 6.8 ± 0.0 6.8 ± 0.1 6.9 ± 0.1 (7.0) (7.0) (6.9) (6.9)200 mM Citrate 6.8 ± 0.1 6.8 ± 0.0 6.7 ± 0.1 6.6 ± 0.2 (7.0) (7.0) (6.9)(6.9) Base Formulation: 60% NAF, 10% Sucrose, 1% Gelatin 2% Arginine

TABLE 42 Formulation 0.5 mM 1.0 mM 2.0 mM 5.0 mM 10 mM Ingredients NoEDTA EDTA EDTA EDTA EDTA EDTA KPO4 buffer, 100 mM 100 mM 100 mM 100 mM100 mM 100 mM pH 7.2 (1.1 mM from virus included) Sucrose (0.7% 10%   10%   10%    10%   10%   10% from virus included) Gelatin  1%    1%   1%    1%    1%    1% Arginine  2%    2%    2%    2%    2%    2% EDTA 0% 0.0186% 0.037% 0.0744% 0.186% 0.372% (0.5 mM) (1.0 mM) (2.0 mM) (5.0mM) (10 mM) NAF (from 10%    10%   10%    10%   10%   10% virus:B/Hongkong; A/Panama) NAF (added) 50%    50%   50%    50%   50%   50% 1NKOH or 1N titrate titrate titrate titrate titrate titrate HCl to pH 7.2to pH 7.2 to pH 7.2 to pH 7.2 to pH 7.2 to pH 7.2 to pH 7.2 PurifiedWater q.s. q.s. q.s. q.s. q.s. q.s.

TABLE 43 A/Panama A/Panama B/Hongkong B/Hongkong Aliquot 1 Aliquot 2Aliquot 1 Aliquot 2 A/Panama, Starting material Ave. of 9 Ave. of 6plates plates 8.1 ± 0.2 7.9 ± 0.1 (8.5) (7.9) Pre-diluted StartingMaterial (1:10) 8.0 ± 0.1 none n/a n/a (8.0) 0% EDTA 6.1 ± 0.2 6.1 ± 0.26.7 ± 0.1 6.7 ± 0.1 (7.0) (7.0) (6.9) (6.9) 0.5 mM EDTA 6.3 ± 0.2 6.3 ±0.1 6.7 ± 0.1 6.6 ± 0.1 (7.0) (7.0) (6.9) (6.9) 1.0 mM EDTA 6.5 ± 0.16.5 ± 0.1 6.8 ± 0.1 6.6 ± 0.1 (7.0) (7.0) (6.9) (6.9) 2.0 mM EDTA 6.6 ±0.0 6.8 ± 0.1 6.6 ± 6.6 ± 0.1 (7.0) (7.0) (6.9) (6.9) 5.0 mM EDTA 6.7 ±0.0 6.8 ± 0.1 6.6 ± 0.1 6.7 ± 0.2 (7.0) (7.0) (6.9) (6.9)  10 mM EDTA6.8 ± 0.1 6.7 ± 0.1 6.7 ± 0.1 6.6 ± 0.2 (7.0) (7.0) (6.9) (6.9) BaseFormulation: 100 mM KPO4, 60% NAF, 10% Sucrose, 1% Gelatin 2% Arginine

TABLE 44 Ingredients Liq28 Liq29 Liq30 KPO4 buffer, pH 7.2 100 mM 100 mM(1.1 mM from virus included) Citrate buffer, pH 7.2 n/a n/a 100 mMSucrose (0.7% from   10%   10%   10% virus included) Gelatin    2% n/a   2% Arginine    2%    2%    2% EDTA (5 mM) (10 mM) (10 mM) 0.186%0.372% 0.372% NAF (from virus)   10%   10%   10% NAF (added)   50%   50%  50% 1N HCl or titrate titrate titrate 1N KOH to pH 7.2 to pH 7.2 to pH7.2 to pH 7.2 Purified Water q.s. q.s. q.s.

TABLE 45 A SP stabilized B/Hongkong Target: 6.9 15° C. (weekly) 0 0 2 24 4 6 6 8 8 10 10 12 12 14 14 Formulation Liq 26 6.8 6.8 6.6 6.4 6.3 6.35.8 6.0 5.4 5.5 5.4 5.6 5.3 5.4 5.3 5.3 10Suc/2Gel/2Arg/10 mM EDTA Liq26a (deg) 6.8 6.7 6.5 6.5 6.5 6.3 10Suc/2Gel/2Arg/10 mM EDTA Liq 28 6.25.9 5.8 5.9 6.2 6.0 5.4 5.3 5.1 5.1 disc. 10Suc/2Gel/2Arg/5 mM EDTA Liq28a 6.5 6.5 6.2 6.0 6.0 5.9 5.8 5.6 disc. 10Suc/2Gel/2Arg/5 mM EDTA Liq29 4.9 4.9 4.8 4.7 5.0 4.8 4.2 4.0 ud 3.6 disc. 10Suc/2Arg/10 mM EDTALiq 29a 6.3 6.4 6.0 6.3 6.1 5.9 5.8 5.7 disc. 10Suc/2Arg/10 mM EDTA Liq30 6.6 6.6 6.3 6.4 6.7 6.5 5.9 5.9 5.4 5.4 disc.Citr./10Suc/2Gel/2Arg/10 mM EDTA Liq 30a 6.6 6.6 6.1 6.2 6.1 5.9 5.8 5.8disc. Citr./10Suc/2Gel/2Arg/10 mM EDTA B SP stabilized B/HongkongTarget: 6.9 4° C. (Monthly) 0 0 1 1 2 2 3 3 4 4 Formulation Liq 26 6.86.8 6.6 6.7 6.6 6.5 6.6 6.6 6.3 10Suc/2Gel/2Arg/10 mM EDTA Liq 26a (deg)6.8 6.7 6.9 6.9 10Suc/2Gel/2Arg/10 mM EDTA Liq 28 6.2 5.9 6.6 6.4 6.66.5 5.8 5.8 10Suc/2Gel/2Arg/5 mM EDTA Liq 28a 6.5 6.5 6.7 6.8 6.5 6.610Suc/2Gel/2Arg/5 mM EDTA Liq 29 4.9 4.9 5.1 5.1 4.9 4.9 disc.10Suc/2Arg/10 mMEDTA Liq 29a 6.3 6.4 6.4 6.5 6.3 6.4 10Suc/2Arg/10 mMEDTA Liq 30 6.6 6.6 6.8 7.1 6.6 6.5 6.2 6.0 Citr./10Suc/2Gel/2Arg/10 mMEDTA Liq 30a 6.6 6.6 6.9 6.7 6.4 6.0 Citr./10Suc/2Gel/2Arg/10 mM EDTA CSP stabilized A/Panama Target: 7.5 15° C. (weekly) 0 0 2 2 4 4 6 6 8 810 10 12 12 14 14 Formulation Liq 26 7.4 7.3 7.2 7.1 ud(repd) ud(repd)6.9 6.9 6.7 6.7 6.7 6.9 6.5 6.4 6.4 6.4 10Suc/2Gel/2Arg/10 mM EDTA Liq26a 7.1 7.2 7 7.1 7.1 6.9 6.4 6.8 10Suc/2Gel/2Arg/10 mM EDTA (deg) Liq28 7.5 7.4 7.1 7.1 6.9 7.0 6.7 6.7 6.5 6.5 6.6 6.6 6.1 6.110Suc/2Gel/2Arg/5 mM EDTA Liq 29 7.3 7.3 6.9 6.9 7.0 7.0 6.7 6.8 6.6 6.76.5 6.7 6.0 6.0 10Suc/2Arg/10 mM EDTA Liq 30 7.4 7.3 7.0 7.0 6.8 6.9 6.36.3 6.1 6.3 6.2 6.1 5.8 5.6 Citr./10Suc/2Gel/2Arg/10 mM EDTA D SPstabilized A/Panama Target: 7.5 4° C. (Monthly) 0 0 1 1 2 2 3 3 4 4Formulation Liq 26 7.4 7.3 7.3 7.3 7.3 7.3 7.4 7.3 7.0 7.110Suc/2Gel/2Arg/10 mM EDTA Liq 26a (deg) 7.1 7.2 7.4 7.5 7.1 7.010Suc/2Gel/2Arg/10 mM EDTA Liq 28 7.5 7.4 7.3 7.3 7.1 7.210Suc/2Gel/2Arg/5 mM EDTA Liq 29 7.3 7.3 7.2 7.5 7.1 7.1 10Suc/2Arg/10mM EDTA Liq 30 7.4 7.3 7.3 7.4 7.0 7.2 Citr./10Suc/2Gel/2Arg/10 mM EDTA

TABLE 46 A Ingredients Liq36 Liq37 Liq38 Liq39 Liq40 Liq41 Liq42 Liq43Liq44 Liq45 Liq46 Liq47 KPO4 buffer, 50 mM  50 mM 50 mM 50 mM  50 mM 50mM 50 mM 50 mM 50 mM 50 mM 50 mM  50 mM pH 7.2 (1.1 mM from virusincluded) Sucrose (0.7% 0.0% 7.5% 7.5% 7.5% 7.5% 7.5% 7.5% 10% 10% 10%10% 10% from virus included) Gelatin   0%   0%   0%   1%   1%   2%   2% 0%  0%  0%  1%  1% Arginine   2%   2%   4%   0%   4%   0%   2%  4%  2% 4%  2%  2% EDTA  1 mM 2.7 mM  5 mM  1 mM 2.7 mM  5 mM  1 mM  5 mM  5 mM 1 mM  1 mM 2.7 mM NAF (from  10%  10%  10%  10%  10%  10%  10% 10% 10%10% 10% 10% virus: B/Hongkong; A/Panama) NAF (added)  50%  50%  50%  50% 50%  50%  50% 50% 50% 50% 50% 50% 1N KOH or 1N titrate titrate titratetitrate titrate titrate titrate titrate titrate titrate titrate titrateHCl to pH 7.2 to pH to pH to pH to pH to pH to pH to pH to pH to pH topH to pH to pH 7.2 7.2 7.2 7.2 7.2 7.2 7.2 7.2 7.2 7.2 7.2 7.2 PurifiedWater q.s. q.s q.s q.s q.s q.s q.s q.s q.s q.s q.s q.s B IngredientsLiq48 Liq49 Liq50 Liq51 Liq52 Liq53 Liq54 Liq55 Liq56 Liq57 Liq58 Liq59KPO4 buffer, 50 mM 50 mM  50 mM  50 mM  50 mM 50 mM  50 mM  50 mM 50 mM50 mM 50 mM 50 mM pH 7.2 (1.1 mM from virus included) Sucrose (0.7% 10%10% 10% 10% 15% 15% 15% 15% 15% 15% 15% 15% from virus included) Gelatin 1%  2%  2%  2%  0%  0%  0%  1%  1%  1%  2%  2% Arginine  4%  0%  0%  4% 2%  2%  4%  0%  0%  4%  2%  4% EDTA  5 mM  1 mM 2.7 mM 2.7 mM 2.7 mM  1mM 2.7 mM 2.7 mM  5 mM  1 mM  5 mM  1 mM NAF (from 10% 10% 10% 10% 10%10% 10% 10% 10% 10% 10% 10% virus: B/Hongkong; A/Panama) NAF (added) 50%50% 50% 50% 50% 50% 50% 50% 50% 50% 50% 50% 1N KOH or 1N titrate titratetitrate titrate titrate titrate titrate titrate titrate titrate titratetitrate HCl to pH 7.2 to pH to pH to pH to pH to pH to pH to pH to pH topH to pH to pH to pH 7.2 7.2 7.2 7.2 7.2 7.2 7.2 7.2 7.2 7.2 7.2 7.2Purified Water q.s. q.s q.s q.s q.s q.s q.s q.s q.s q.s q.s q.s

TABLE 47 SP stabilized B/Hongkong Target: 6.9 4° C. (monthly) 0 0 0 1 11 2 2 2 3 3 3 4 4 4 5 5 5 Liq 36 6.8 6.8 6.7 50 mM KPO4/0Suc/0Gel/2Arg/1mm EDTA Liq 37 6.6 6.6 6.7 50 mM KPO4/7.5Suc/0Gel/2Arg/2.7 mm EDTA Liq38 6.6 6.5 6.6 50 mM KPO4/7.5Suc/0Gel/4Arg/5 mm EDTA Liq 39 6.9 6.8 6.750 mM KPO4/7.5Suc/1Gel/0Arg/1 mm EDTA Liq 40 6.7 6.7 6.7 50 mMKPO4/7.5Suc/1Gel/4Arg/2.7 mm EDTA Liq 41 7.0 7.0 7.0 50 mMKPO4/7.5Suc/2Gel/0Arg/5 mm EDTA Liq 42 6.9 6.9 7.0 50 mMKPO4/7.5Suc/2Gel/2Arg/1 mm EDTA Liq 43 6.8 6.8 6.7 50 mMKPO4/10Suc/0Gel/4Arg/5 mm EDTA Liq 44 6.8 6.9 6.9 50 mMKPO4/10Suc/0Gel/2Arg/5 mm EDTA Liq 45 6.7 6.8 6.8 50 mMKPO4/10Suc/0Gel/4Arg/1 mm EDTA Liq 46 7.0 7.0 6.8 50 mMKPO4/10Suc/1Gel/2Arg/1 mm EDTA Liq 47 7.1 7.0 7.0 50 mMKPO4/10Suc/1Gel/2Arg/2.7 mm EDTA Liq 48 7.0 6.9 6.9 50 mMKPO4/10Suc/1Gel/4Arg/5 mm EDTA Liq 49 7.0 7.0 7.0 50 mMKPO4/10Suc/2Gel/0Arg/1 mm EDTA Liq 50 7.0 6.8 7.0 50 mMKPO4/10Suc/2Gel/0Arg/2.7 mm EDTA Liq 51 6.9 6.8 6.9 50 mMKPO4/10Suc/2Gel/4Arg/2.7 mm EDTA Liq 52 6.6 6.7 6.7 50 mMKPO4/15Suc/0Gel/2Arg/2.7 mm EDTA Liq 53 6.7 6.8 6.7 50 mMKPO4/15Suc/0Gel/2Arg/1 mm EDTA Liq 54 6.7 6.8 6.7 50 mMKPO4/15Suc/0Gel/4Arg/2.7 mm EDTA Liq 55 6.9 6.8 6.9 50 mMKPO4/15Suc/1Gel/0Arg/2.7 mm EDTA Liq 56 6.8 6.8 7   50 mMKPO4/15Suc/1Gel/0Arg/5 mm EDTA Liq 57 6.9 6.8 6.8 50 mMKPO4/15Suc/1Gel/4Arg/1 mm EDTA Liq 58 6.9 6.8 6.9 50 mMKPO4/15Suc/2Gel/2Arg/5 mm EDTA Liq 59 50 mM KPO4/15Suc/2Gel/4Arg/1 mmEDTA

TABLE 48 SP stabilized A/Panama Target: 7.0 4° C. (monthly) 0 0 0 1 1 12 2 2 3 3 3 4 4 4 5 5 5 Liq 36 6.4 6.2 6.1 50 mM KPO4/0Suc/0Gel/2Arg/1mm EDTA Liq 37 6.6 6.7 6.6 50 mM KPO4/7.5Suc/0Gel/2Arg/2.7 mm EDTA Liq38 6.7 6.7 6.6 50 mM KPO4/7.5Suc/0Gel/4Arg/5 mm EDTA Liq 39 6.4 6.4 6.450 mM KPO4/7.5Suc/1Gel/0Arg/1 mm EDTA Liq 40 6.7 6.7 6.6 50 mMKPO4/7.5Suc/1Gel/4Arg/2.7 mm EDTA Liq 41 6.0 6.0 6.0 50 mMKPO4/7.5Suc/2Gel/0Arg/5 mm EDTA Liq 42 5.9 5.9 6.0 50 mMKPO4/7.5Suc/2Gel/2Arg/1 mm EDTA Liq 43 6.5 6.6 6.5 50 mMKPO4/10Suc/0Gel/4Arg/5 mm EDTA Liq 44 6.5 6.4 6.4 50 mMKPO4/10Suc/0Gel/2Arg/5 mm EDTA Liq 45 6.3 6.3 6.4 50 mMKPO4/10Suc/0Gel/4Arg/1 mm EDTA Liq 46 6.3 6.3 6.3 50 mMKPO4/10Suc/1Gel/2Arg/1 mm EDTA Liq 47 6.7 6.7 6.6 50 mMKPO4/10Suc/1Gel/2Arg/2.7 mm EDTA Liq 48 6.6 6.6 6.7 50 mMKPO4/10Suc/1Gel/4Arg/5 mm EDTA Liq 49 6.1 6.1 6.1 50 mMKPO4/10Suc/2Gel/0Arg/1 mm EDTA Liq 50 6.6 6.6 6.5 50 mMKPO4/10Suc/2Gel/0Arg/2.7 mm EDTA Liq 51 6.9 7.0 6.9 50 mMKPO4/10Suc/2Gel/4Arg/2.7 mm EDTA Liq 52 6.8 6.7 6.8 50 mMKPO4/15Suc/0Gel/2Arg/2.7 mm EDTA Liq 53 6.7 6.7 6.7 50 mMKPO4/15Suc/0Gel/2Arg/1 mm EDTA Liq 54 6.9 6.9 6.8 50 mMKPO4/15Suc/0Gel/4Arg/2.7 mm EDTA Liq 55 6.7 6.9 6.7 50 mMKPO4/15Suc/1Gel/0Arg/2.7 mm EDTA Liq 56 6.8 6.6 6.8 50 mMKPO4/15Suc/1Gel/0Arg/5 mm EDTA Liq 57 6.9 6.8 6.9 50 mMKPO4/15Suc/1Gel/4Arg/1 mm EDTA Liq 58 6.8 6.8 7.0 50 mMKPO4/15Suc/2Gel/2Arg/5 mm EDTA Liq 59 6.9 6.8 6.6 50 mMKPO4/15Suc/2Gel/4Arg/1 mm EDTA

TABLE 49 Formulations in the Comparison Study purified Unpurified VH VH1 DiaFiltered VH 2 10% AF 60% AF <1% AF 10% AF 60% AF <1% AF  7%SucrosePO₄ (base stabilizers) 2 3 1 16 Wyeth Clinical Formulation 7 8 45 6  7% SucrosePO₄ Arg 17  7% SucrosePO₄ Arg, Gel 18 10% SucrosePO₄,Arg, Gel 9 10% SucrosePO₄, Arg 10 10% SucrosePO₄, Arg, PVP 12 10%SucrosePO₄, Arg, Dextran 11 10% SucrosePO₄, Arg, Gel, EDTA 13 10%Sucrose, Arg, Gel, Histidine 14 10% Sucrose, Arg, Gel, Hist., EDTA 15

TABLE 50 Stability of Purified VH vs. Unpurified VH (FluMist): when bothare stabilized by the purified formulation Stability slope @40° C.(±SE), at six months Purified formulation FluMist* (Log FFU/month) (LogFFU/month) A/NC −0.020 ± 0.027 −0.035 ± 0.016 A/Pan −0.011 ± 0.020−0.079 ± 0.035 B/HK −0.138 ± 0.022 −0.151 ± 0.018 Formulation: 7%Sucrose, 1% gelatin, 1% arginine [‘7/1/1’ formulation] *60% AF level

TABLE 51 Stability of purified VH vs. FluMist: When FluMist isstabilized by ‘10/2/2’ formulation Stability slope @4° C. Initial (±SE),at six Potency loss months [log FFU/month] (Log FFU)** Purified PurifiedVH FluMist* VH FluMist* A/NC −0.020 ± 0.027 −0.011 ± 0.019 0.4 0.5 A/Pan−0.011 ± 0.020 −0.093 ± 0.032 0.9 0.9 B/HK −0.138 ± .022  −0.107 ± 0.0250 0.3 Purified VH formulation: 7% Sucrose, 1% gelatin, 1% arginine[‘7/1/1’ formulation], no added AF *FluMist formulation: 10% Sucrose, 2%gelatin, 2% arginine [‘10/2/2’ formulation], 60% AF level **Based onlinear regression

TABLE 52 Stability of purified VH formulaiton vs. FluMist: When FluMistis stabilized by ‘10/2/2 Histidine’ formulation Stability slope @40° C.Initial (±SE), at six Potency loss** months [log FFU/month] (log FFU)Purified Purified VH FluMist* VH FluMist* A/NC −0.020 ± 0.027 −0.068 ±0.014 0.4 0 A/Pan −0.011 ± 0.020 −0.072 ± 0.012 0.9 0 B/HK −0.138 ±.022  −0.061 ± 0.020 0 0 Purified VH formulation: 7% Sucrose, 1%gelatin, 1% arginine [‘7/1/1’ formulation], no added AF *FluMistformulation: Histidine, 10% Sucrose, 2% gelatin, 2% arginine [‘10/2/2His’ formulation], 60% AF level **Based on linear regression

TABLE 53 Comparison of Method Performance Method Performance TestParameter Manual Semi-automated Precision/Between SD range from 0.07 toSD range from 0.06–0.09 Test Variability 0.11 log TCID₅₀ units¹log₁₀TCID₅₀/mL^(2,3) (SD)) Linearity Passes test for lack of Passes testfor lack of fit to a linear model at fit to a linear model a the 1%significance t the 1% significance level. level.³ Accuracy Slope range0.986–1.007. Slopes range 1.00–1.02.³ Range 4.7–9.5 4.2–9.3log₁₀TCID₅₀/mL. log₁₀TCID₅₀/mL.³ ¹Between-test SD from 9 tests on thesame material (each test results is an average of 12 determinations over3 days), by the same analyst group, on the same pipetting station. Thematerials tested include 3 independent manufacturing lots of each ofthree virus strains (H1N1, H3N2 and B). ²Between-test SD from 6 tests onthe same material (each test results is an average of 12 determinationsover 3 days), by the same analyst group, on the same pipetting station.The materials tested include one lot of each of three virus strains(H1N1, H3N2 and B). ³Validation Report for Semi-Automated TCID50 PotencyAssay for Influenza Virus Monovalent.

TABLE 54 Inter-Assay Comparison Mean Titer (log₁₀TCID₅₀/mL) Semi- ManualAutomated Differ- 90% CI Strain Assay Assay ence (LB, UB)A/NewCaledonia/20/99 9.40 9.42 −0.02 (−0.05, 0.01) B/Yamanashi/166/988.47 8.40 0.07 (0.03, 0.10)

TABLE 55 Manual “gold SemiAutomated standard” readout MTT readoutCPE-positive CPE-negative CPE-positive TP FP (A₅₇₀ ≦ cutoff)CPE-negative FN TN (A₅₇₀ > cutoff) All positives All negatives

TABLE 56 SemiAutomated TCID₅₀ Potency Assay for Influenza VirusMonovalent: Sensitivity and Specificity Estimates Based on the “GoldStandard” Validated Manual CPE Readout and the MTT Assay A570 CutoffValue of 0.5254 True False Sensi- True False Speci- positive negativetivity^(a) negative positive ficity^(b) AB 7,091 61 99.15%  7,247 199.99% (N = 14,400) QC 15,835 301 98.13% 15,106 198 98.71% (N = 31,440)AB and QC 22,926 362 98.45% 22,353 199 99.12% (N = 45,840) Control17,248 167 99.05% 15,882 3 99.99% ATR.0126 ASENSITIVITY = (TRUEPOSITIVE)/ALL POSITIVE BSPECIFICITY = (TRUE NEGATIVE)/ALL NEGATIVE

TABLE 57 Control Well (CPE-negative) Absorbance Values Obtained by thetwo groups with previous Values Reported Shown for Comparison (previousCombined values) 2^(nd) group 1^(st) group (two groups) Control Wellcount 2880 6288 9168 6720 A₅₇₀Average 1.226 1.235 1.231 1.261 SD 0.170.20 0.19 0.15

TABLE 58 Instrument-to-Instrument Comparison: SemiAutomated TCID₅₀Potency Values for Reference Virus Strains Reference Virus Strain (Meanlog₁₀TCID₅₀/mL ± SD) Instrument A/New Caledonia/ A/Sydney/ B/Yamanashi/(group) 20/99 05/97 166/98 AZ-039 (1^(st))^(1,2) 9.2 ± 0.15 8.6 ± 0.098.4 ± 0.10 AZ-040 (1^(st))^(1,3) 9.3 ± 0.08 8.6 ± 0.01 8.4 ± 0.10 AZ-036(2^(nd))¹ 9.2 ± 0.08 8.5 ± 0.05 8.3 ± 0.06 ¹Number of tests (AZ-036, N =9; AZ-039, N = 5; AZ-040, N = 2); 1^(st) = first group, 2^(nd) = secondgroup. ²For AZ-039, one test result rejected due to failure of intra-daySD acceptance criteria ³For AZ-040, four test results rejected due tofailure of intra-day SD acceptance criteria or mishandling of plates

TABLE 59 Analyst-to-Analyst Comparison: SemiAutomated TCID₅₀ PotencyValues for Reference Virus Strains Using Instruments AZ-039 or AZ-036Reference Virus Strain (Mean log₁₀TCID₅₀/mL ± SD)^(a) A/New A/ B/Caledonia/20/99 Sydney/05/97 Yamanashi/166/98 first group AZ-039 AZ-039AZ-039 Analyst # 1 9.3 ± 0.19 8.5 ± 0.25 8.4 ± 0.26 Analyst # 2 9.1 ±0.17 8.5 ± 0.27 8.4 ± 0.16 Analyst # 3 9.1 ± 0.16 8.5 ± 0.15 8.4 ± 0.19Analyst # 4 9.2 ± 0.24 8.6 ± 0.21 8.6 ± 0.24 Analyst # 5 9.1 ± 0.21 8.6± 0.19 8.3 ± 0.23 Analyst # 6 9.4 ± 0.21 8.7 ± 0.20 8.6 ± 0.21 secondgroup AZ-036 AZ-036 AZ-036 Analyst # 7 9.4 ± 0.16 8.5 ± 0.21 8.3 ± 0.18Analyst # 8 9.2 ± 0.21 8.5 ± 0.18 8.2 ± 0.15 Analyst # 9 9.3 ± 0.16 8.5± 0.20 8.3 ± 0.16 ^(a)Mean potency values are derived from fourreplicates obtained over three test days (n = 12).

1. A method of making an influenza virus composition, the method comprising the steps of: a. passaging an influenza virus through eggs; b. chilling the eggs to a temperature of between about 2° C. to about 8° C.; c. harvesting the influenza virus; d. warming the influenza virus to a temperature from about 28° C. to 40° C.; and, e. filtering the influenza virus through a membrane; wherein warming occurs before and/or during filtering of the influenza virus.
 2. The method of claim 1, wherein the influenza virus composition comprises one or more influenza A virus strain and/or one or more influenza B virus strain.
 3. The method of claim 1, wherein the influenza virus is filtered through a microfilter, wherein the microfilter has a pore size of from about 0.2 micrometer to about 0.45 micrometer.
 4. The method of claim 1, wherein the influenza virus is warmed to a temperature of about 31° C.
 5. The method of claim 1, wherein the influenza virus is warmed to a temperature from about 28° C. to about 36° C.
 6. The method of claim 1, wherein the influenza virus is warmed to a temperature from about 28° C. to about 34° C.
 7. The method of claim 1, wherein the influenza virus is warmed to a temperature from about 30° C. to about 33° C.
 8. The method of claim 1, wherein warming occurs before filtering of the influenza virus.
 9. The method of claim 1, wherein warming occurs during the filtering of the influenza virus.
 10. The method of claim 1, wherein warming occurs both before and during the filtering of the influenza virus.
 11. The method of claim 1, wherein warming is performed for a duration of about 1 hour.
 12. The method of claim 1, wherein warming is performed for a duration of from about 30 minutes to about 240 minutes.
 13. The method of claim 12, wherein warming is performed for a duration of from about 45 minutes to about 200 minutes.
 14. The method of claim 12, wherein warming is performed for a duration of from about 60 minutes to about 90 minutes.
 15. The method of claim 1, wherein the eggs are rocked during said passage.
 16. The method of claim 15, wherein rocking comprises tilting the eggs at rate of approximately 1 cycle per minute or less, about 5 cycles per minute or less, or about 10 cycles per minute or less.
 17. The method of claim 15, wherein the eggs are rocked for approximately 12 hours.
 18. The method of claim 15, wherein the eggs are rocked for approximately 24–48 hours.
 19. The method of claim 15, further comprising a secondary incubation.
 20. The method of claim 19, wherein the eggs are rocked during the secondary incubation.
 21. The method of claim 15, wherein the influenza virus composition comprises one or more influenza A virus strain and/or one or more influenza B virus strain.
 22. The method of claim 15, wherein a TCID₅₀ of the influenza virus passaged through rocked eggs is at least 0.4 log greater than a TCID₅₀ of the same influenza virus passaged through non-rocked eggs.
 23. The method of claim 1, 2, 3, 4, 5, 6 or 7, wherein the influenza virus composition has a filtration potency loss of less than 0.3 log TCID/ml.
 24. The method of claim 1, wherein the passaging an influenza virus through eggs takes place at a temperature less than or equal to 35° C.
 25. The method of claim 1, 5, 6 or 7, wherein the influenza virus composition has a filtration potency loss of less than 0.2 log TCID/ml.
 26. The method of claim 1, 5, 6 or 7, wherein the influenza virus composition has a filtration potency loss of less than 0.1 log TCID/ml.
 27. The method of claim 1, 5, 6 or 7, wherein the influenza virus composition has no filtration potency loss. 